Production of carotenoids in oleaginous yeast and fungi

ABSTRACT

The present invention provides systems for producing engineered oleaginous yeast or fungi that express carotenoids

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/903,938, filed Oct. 13, 2010, which is a divisional application of application Ser. No. 11/385,580, filed Mar. 20, 2006, now U.S. Pat. No. 7,851,199, which claims the benefit of U.S. Provisional Application No. 60/663,621, filed Mar. 18, 2005, all of which are hereby incorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Carotenoids are organic pigments ranging in color from yellow to red that are naturally produced by certain organisms, including photosynthetic organisms (e.g., plants, algae, cyanobacteria), and some fungi. Carotenoids are responsible for the orange color of carrots, as well as the pink in flamingos and salmon, and the red in lobsters and shrimp. Animals, however, cannot produce carotenoids and must receive them through their diet.

Carotenoid pigments (e.g., β-carotene and astaxanthin) are used industrially as ingredients for food and feed stocks, both serving a nutritional function and enhancing consumer acceptability. For example, astaxanthin is widely used in salmon aquaculture to provide the orange coloration characteristic of their wild counterparts. Some carotenoids are also precursors of vitamin A. Also, carotenoids have antioxidant properties, and may have various health benefits (see, for example, Jyonouchi et al., Nutr. Cancer 16:93, 1991; Giovannucci et al., J. Natl. Cancer Inst. 87:1767, 1995; Miki, Pure Appi. Chem. 63:141, 1991; Chew et al., Anticancer Res. 19:1849, 1999; Wang et al., Antimicrob. Agents Chemother. 44:2452, 2000). Some carotenoids such as β-carotene, lycopene, and lutein are currently sold as nutritional supplements.

In general, the biological systems that produce carotenoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practicable. Thus, most carotenoids used in industry are produced by chemical synthesis. There is a need for improved biological systems that produce carotenoids. Some efforts have previously been made to genetically engineer certain bacteria or fungi to produce higher levels of carotenoids (see, for example, Misawa et al., J. Biotechnol. 59:169, 1998; Visser et al., FEMS Yeast Research 4:221, 2003). However, improved systems, allowing higher levels of production and greater ease of isolation, are needed.

SUMMARY OF THE INVENTION

The present invention provides improved systems for the biological production of carotenoids. In one aspect, the invention encompasses the discovery that it is desirable to produce carotenoids in oleaginous organisms. Without wishing to be bound by any particular theory, the present inventors propose that biological systems may be able to accumulate higher levels of carotenoids if the compounds are sequestered in lipid bodies. Regardless of whether absolute levels are higher, however, carotenoids that are accumulated within lipid bodies in oleaginous organisms are readily isolatable through isolation of the lipid bodies.

The present invention therefore provides oleaginous fungi (including, for example, yeast or other unicellular fungi) that produce one or more carotenoids. The present invention also provides methods of constructing such yeast and fungi, methods of using such yeast and fungi to produce carotenoids, and methods of preparing carotenoid-containing compositions, such as food or feed additives, or nutritional supplements, using carotenoids produced in such oleaginous yeast or fungi. In particular, the present invention provides systems and methods for generating yeast and fungi containing one or more oleaginic and/or carotenogenic modifications that increase the oleaginicity and/or alter their carotenoid-producing capabilities as compared with otherwise identical organisms that lack the modification(s).

The present invention further encompasses the general recognition that lipid-accumulating systems are useful for the production and/or isolation of lipophilic agents (such as, but not limited to isoprenoids, or isoprenoid-derived compounds). Thus, according to the present invention, it is desirable to engineer organisms to produce such lipophilic agents and/or to accumulate lipid.

Various other aspects of the present invention will be apparent to those of ordinary skill in the art from the present description, including the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1D depicts certain common carotenoids.

FIG. 2 depicts how sufficient levels of acetyl-CoA and NADPH may be accumulated in the cytosol of oleaginous organisms to allow for production of significant levels of cytosolic lipids. Enzymes: 1, pyruvate decarboxylase; 2, malate dehydrogenase; 3, malic enzyme; 4, pyruvate dehydrogenase; 5, citrate synthase; 6, ATP-citrate lyase; 7, citrate/malate translocase.

FIGS. 3A and 3B depict the mevalonate isoprenoid biosynthesis pathway, which typically operates in eukaryotes, including fungi.

FIG. 4 depicts the mevalonate-independent isoprenoid biosynthesis pathway, also known as the DXP pathway, which typically operates in bacteria and in the plastids of plants.

FIG. 5 depicts intermediates in the isoprenoid biosynthesis pathway and how they feed into biosynthetic pathways of other biomolecules, including carotenoids as well as non-carotenoid compounds such as sterols, steroids, and vitamins, such as vitamin E or vitamin K.

FIGS. 6A-6D illustrate various carotenoid biosynthetic pathways. FIG. 6A highlights branches leading to various cyclic and acyclic xanthophylls; FIG. 6B shows certain X. dendrorhous pathways that generate dicyclic and monocyclic carotenoids, including astaxanthin; FIG. 6C shows interconnecting pathways for converting β-carotene into any of a variety of other carotenoids, including astaxanthin; FIG. 6D depicts possible routes of synthesis of cyclic carotenoids and common plant and algal xanthophylls from neurosporene.

FIG. 7 and subparts FIGS. 7A-7I show an alignment of certain representative fungal HMG-CoA reductase polypeptides. As can be seen, these polypeptides show very high identity across the catalytic region, and also have complex membrane spanning domains. In some embodiments of the invention, these membrane-spanning domains are disrupted or are removed, so that, for example, a hyperactive version of the polypeptide may be produced.

FIGS. 8A-8D depict schematic representations of plasmids generated and described in detail in the exemplification.

DEFINITIONS

Carotenogenic modification: The term “carotenogenic modification”, as used herein, refers to a modification of a host organism that adjusts production of one or more carotenoids, as described herein. For example, a carotenogenic modification may increase the production level of one or more carotenoids, and/or may alter relative production levels of different carotenoids. In principle, an inventive carotenogenic modification may be any chemical, physiological, genetic, or other modification that appropriately alters production of one or more carotenoids in a host organism produced by that organism as compared with the level produced in an otherwise identical organism not subject to the same modification. In most embodiments, however, the carotenogenic modification will comprise a genetic modification, typically resulting in increased production of one or more selected carotenoids. In some embodiments, the selected carotenoid is one or more of astaxanthin, β-carotene, canthaxanthin, lutein, lycopene, phytoene, zeaxanthin, and/or modifications of zeaxanthin or astaxanthin (e.g., glucoside, esterified zeaxanthin or astaxanthin). In some embodiments, the selected carotenoid is one or more xanthophylls, and/or a modification thereof (e.g., glucoside, esterified xanthophylls). In certain embodiments, the selected xanthophyl is selected from the group consisting of astaxanthin, lutein, zeaxanthin, lycopene, and modifications thereof. In some embodiments, the selected carotenoid is one or more of astaxanthin, β-carotene, canthaxanthin, lutein, lycopene, and zeaxanthin and/or modifications of zeaxanthin or astaxanthin. In some embodiments, the carotenoid is β-carotene. In some embodiments, the selected carotenoid is astaxanthin. In some embodiments, the selected carotenoid is other than β-carotene.

Carotenogenic polypeptide: The term “carotenogenic polypeptide”, as used herein, refers to any polypeptide that is involved in the process of producing carotenoids in a cell, and may include polypeptides that are involved in processes other than carotenoid production but whose activities affect the extent or level of production of one or more carotenoids, for example by scavenging a substrate or reactant utilized by a carotenoid polypeptide that is directly involved in carotenoid production. Carotenogenic polypeptides include isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, and isoprenoid biosynthesis competitor polypeptides, as those terms are defined herein. The term also encompasses polypeptides that may affect the extent to which carotenoids are accumulated in lipid bodies.

Carotenoid: The term “carotenoid” is understood in the art to refer to a structurally diverse class of pigments derived from isoprenoid pathway intermediates. The commitment step in carotenoid biosynthesis is the formation of phytoene from geranylgeranyl pyrophosphate. Carotenoids can be acyclic or cyclic, and may or may not contain oxygen, so that the term carotenoids include both carotenes and xanthophylls. In general, carotenoids are hydrocarbon compounds having a conjugated polyene carbon skeleton formally derived from the five-carbon compound IPP, including triterpenes (C₃₀ diapocarotenoids) and tetraterpenes (C₄₀ carotenoids) as well as their oxygenated derivatives and other compounds that are, for example, C₃₅, C₅₀, C₆₀, C₇₀, C₈₀ in length or other lengths. Many carotenoids have strong light absorbing properties and may range in length in excess of C₂₀₀. C₃₀ diapocarotenoids typically consist of six isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship. Such C₃₀ carotenoids may be formally derived from the acyclic C₃₀H₄₂ structure, having a long central chain of conjugated double bonds, by: (i) hydrogenation (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes. C₄₀ carotenoids typically consist of eight isoprenoid units joined in such a manner that the arrangement of isoprenoid units is reversed at the center of the molecule so that the two central methyl groups are in a 1,6-positional relationship and the remaining non-terminal methyl groups are in a 1,5-positional relationship. Such C₄₀ carotenoids may be formally derived from the acyclic C₄₀H₅₆ structure, having a long central chain of conjugated double bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes. The class of C₄₀ carotenoids also includes certain compounds that arise from rearrangements of the carbon skeleton, or by the (formal) removal of part of this structure. More than 600 different carotenoids have been identified in nature; certain common carotenoids are depicted in FIG. 1. Carotenoids include but are not limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin, canthaxanthin, capsorubrin, β-cryptoxanthin, α-carotene, β-carotene, β,Ψ-carotene, δ-carotene, ε-carotene, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, γ-carotene, Ψ-carotene, 4-keto-γ-carotene, ζ-carotene, α-cryptoxanthin, deoxyflexixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol, isorenieratene, β-isorenieratene, lactucaxanthin, lutein, lycopene, myxobactone, neoxanthin, neurosporene, hydroxyneurosporene, peridinin, phytoene, rhodopin, rhodopin glucoside, 4-keto-rubixanthin, siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene, 4-keto-torulene, 3-hydroxy-4-keto-torulene, uriolide, uriolide acetate, violaxanthin, zeaxanthin-β-diglucoside, zeaxanthin, and C30 carotenoids. Additionally, carotenoid compounds include derivatives of these molecules, which may include hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups. Further, included carotenoid compounds include ester (e.g., glycoside ester, fatty acid ester) and sulfate derivatives (e.g., esterified xanthophylls).

Carotenoid biosynthesis polypeptide: The term “carotenoid biosynthesis polypeptide” refers to any polypeptide that is involved in the synthesis of one or more carotenoids. To mention but a few, these carotenoid biosynthesis polypeptides include, for example, polypeptides of phytoene synthase, phytoene dehydrogenase (or desaturase), lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase, carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase. Representative examples of carotenoid biosynthesis polypeptide sequences are presented in Tables 17-25.

Gene: The term “gene”, as used herein, generally refers to a nucleic acid encoding a polypeptide, optionally including certain regulatory elements that may affect expression of one or more gene products (i.e., RNA or protein).

Heterologous: The term “heterologous”, as used herein to refer to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is being expressed. It will be understood that, in general, when a heterologous gene or polypeptide is selected for introduction into and/or expression by a host cell, the particular source organism from which the heterologous gene or polypeptide may be selected is not essential to the practice of the present invention. Relevant considerations may include, for example, how closely related the potential source and host organisms are in evolution, or how related the source organism is with other source organisms from which sequences of other relevant polypeptides have been selected.

Host cell: As used herein, the “host cell” is a yeast or fungal cell that is manipulated according to the present invention to accumulate lipid and/or to express one or more carotenoids as described herein. A “modified host cell”, as that term is used herein, is a host cell that contains at least one oleaginic modification and/or at least one carotenogenic modification according to the present invention.

Isolated: The term “isolated”, as used herein, means that the isolated entity has been separated from at least one component with which it was previously associated. When most other components have been removed, the isolated entity is “purified”. Isolation and/or purification may be performed using any techniques known in the art including, for example, fractionation, extraction, precipitation, or other separation.

Isoprenoid biosynthesis competitor polypeptide: The term “isoprenoid biosynthesis competitor polypeptide”, as used herein, refers to a polypeptide whose expression in a cell reduces the level of geranylgeranyl diphosphate (GGPP) available to enter the carotenoid biosynthesis pathway. For example, isoprenoid biosynthesis competitor polypeptides include enzymes that act on isoprenoid intermediates prior to GGPP, such that less GGPP is generated (see, for example, FIG. 5). Squalene synthase is but one isoprenoid biosynthesis competitor polypeptide according to the present invention; representative squalene synthase sequences are presented in Table 16. Prenyldiphosphate synthase enzymes and para-hydroxybenzoate (PHB) polyprenyltransferase are yet additional isoprenoid biosynthesis competitor polypeptides according to the present invention; representative prenyldiphosphate synthase enzymes and PHB polyprenyltransferase polypeptides are presented in Table 29 and 30 respectively.

Isoprenoid biosynthesis polypeptide: The term “isoprenoid biosynthesis polypeptide” refers to any polypeptide that is involved in the synthesis of isoprenoids. For example, as discussed herein, acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, IPP isomerase, FPP synthase, and GGPP synthase, are all involved in the mevalonate pathway for isoprenoid biosynthesis. Each of these proteins is also an isoprenoid biosynthesis polypeptide for purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 7-15.

Isoprenoid pathway: The “isoprenoid pathway” is understood in the art to refer to a metabolic pathway that either produces or utilizes the five-carbon metabolite isopentyl pyrophosphate (IPP). As discussed herein, two different pathways can produce the common isoprenoid precursor IPP—the “mevalonate pathway” and the “non-mevalonate pathway”. The term “isoprenoid pathway” is sufficiently general to encompass both of these types of pathway. Biosynthesis of isoprenoids from IPP occurs by polymerization of several five-carbon isoprene subunits. Isoprenoid metabolites derived from IPP are of varying size and chemical structure, including both cyclic and acyclic molecules. Isoprenoid metabolites include, but are not limited to, monoterpenes, sesquiterpenes, diterpenes, sterols, and polyprenols such as carotenoids.

Oleaginic modification: The term “oleaginic modification”, as used herein, refers to a modification of a host organism that adjusts the desirable oleaginy of that host organism, as described herein. In some cases, the host organism will already be oleaginous in that it will have the ability to accumulate lipid to at least about 20% of its dry cell weight. It may nonetheless be desirable to apply an oleaginic modification to such an organism, in accordance with the present invention, for example to increase (or, in some cases, possibly to decrease) its total lipid accumulation, or to adjust the types or amounts of one or more particular lipids it accumulates (e.g., to increase relative accumulation of triacylglycerol). In other cases, the host organism may be non-oleaginous (though may contain some enzymatic and regulatory components used in other organisms to accumulate lipid), and may require oleaginic modification in order to become oleaginous in accordance with the present invention. The present invention also contemplates application of oleaginic modification to non-oleaginous host strains such that their oleaginicity is increased even though, even after being modified, they may not be oleaginous as defined herein. In principle, the oleaginic modification may be any chemical, physiological, genetic, or other modification that appropriately alters oleaginy of a host organism as compared with an otherwise identical organism not subjected to the oleaginic modification. In most embodiments, however, the oleaginic modification will comprise a genetic modification, typically resulting in increased production and/or activity of one or more oleaginic polypeptides. In some embodiments, the oleaginic modification comprises at least one chemical, physiological, genetic, or other modification; in other embodiments, the oleaginic modification comprises more than one chemical, physiological, genetic, or other modification. In certain aspects where more than one modification is utilized, such modifications can comprise any combination of chemical, physiological, genetic, or other modification (e.g., one or more genetic modification and chemical or physiological modification).

Oleaginic polypeptide: The term “oleaginic polypeptide”, as used herein, refers to any polypeptide that is involved in the process of lipid accumulation in a cell and may include polypeptides that are involved in processes other than lipid biosynthesis but whose activities affect the extent or level of accumulation of one or more lipids, for example by scavenging a substrate or reactant utilized by an oleaginic polypeptide that is directly involved in lipid accumulation. For example, as discussed herein, acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP deaminase, among other proteins, are all involved in lipid accumulation in cells. In general, reducing the activity of pyruvate decarboxylase or isocitrate dehydrogenase, and/or increasing the activity of acetyl CoA carboxylase, ATP-citrate lyase, malic enzyme and/or AMP deaminase is expected to promote oleaginy. Each of these proteins is an oleaginic polypeptide for purposes of the present invention, and sequences of representative examples of these enzymes are provided in Tables 1-6.

Oleaginous: The term “oleaginous”, refers to the ability of an organism to accumulate lipid to at least about 20% of its dry cell weight. In certain embodiments of the invention, oleaginous yeast or fungi accumulate lipid to at least about 25% of their dry cell weight. In other embodiments, inventive oleaginous yeast or fungi accumulate lipid within the range of about 20-45% of their dry cell weight. In some embodiments, oleaginous organisms may accumulate lipid to as much as about 70% of their dry cell weight. In some embodiments of the invention, oleaginous organisms may accumulate a large fraction of total lipid accumulation in the form of triacylglycerol. In certain embodiments, the majority of the accumulated lipid is in the form of triacylglycerol. Alternatively or additionally, the lipid may accumulate in the form of intracellular lipid bodies, or oil bodies. In certain embodiments, the present invention utilizes yeast or fungi that are naturally oleaginous. In some aspects, naturally oleaginous organisms are manipulated (e.g., genetically, chemically, or otherwise) so as to further increase the level of accumulated lipid in the organism. In other embodiments, yeast or fungi that are not naturally oleaginous are manipulated (e.g., genetically, chemically, or otherwise) to accumulate lipid as described herein. For the purposes of the present invention, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and Candida utilis are not naturally oleaginous fungi.

Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. However, the term is also used to refer to specific functional classes of polypeptides, such as, for example, oleaginic polypeptides, carotenogenic polypeptides, isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, and isoprenoid biosynthesis competitor polypeptides. For each such class, the present specification provides several examples of known sequences of such polypeptides. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions (e.g., isocitrate dehydrogenase polypeptides often share a conserved AMP-binding motif; HMG-CoA reductase polypeptides typically include a highly conserved catalytic domain (see, for example, FIG. 7); acetyl coA carboxylase typically has a carboxyl transferase domain; see, for example, Downing et al., Chem. Abs. 93:484, 1980; Gil et al., Cell 41:249, 1985; Jitrapakdee et al. Curr Protein Pept Sci. 4:217, 2003; U.S. Pat. No. 5,349,126, each of which is incorporated herein by reference in its entirety), usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein.

Source organism: The term “source organism”, as used herein, refers to the organism in which a particular polypeptide sequence can be found in nature. Thus, for example, if one or more heterologous polypeptides is/are being expressed in a host organism, the organism in which the polypeptides are expressed in nature (and/or from which their genes were originally cloned) is referred to as the “source organism”. Where more than one heterologous polypeptides are being expressed in a host organism, one or more source organism(s) may be utilized for independent selection of each of the heterologous polypeptide(s). It will be appreciated that any and all organisms that naturally contain relevant polypeptide sequences may be used as source organisms in accordance with the present invention. Representative source organisms include, for example, animal, mammalian, insect, plant, fungal, yeast, algal, bacterial, cyanobacterial, archaebacterial and protozoal source organisms.

Detailed Description of Certain Preferred Embodiments of the Invention

As noted above, the present invention encompasses the discovery that carotenoids can desirably be produced in oleaginous yeast and fungi. According to the present invention, strains that both (i) accumulate lipid, often in the form of cytoplasmic oil bodies and typically to at least about 20% of their dry cell weight; and (ii) produce carotenoid(s) at a level at least about 1%, and in some embodiments at least about 3-20%, of their dry cell weight, are generated through manipulation of host cells (i.e., strains, including, e.g., naturally-occurring strains, strains which have been previously modified, etc.). These manipulated host cells are then used to produce carotenoids, so that carotenoids that partition into the lipid bodies can readily be isolated.

In general, it will be desirable to balance oleaginy and carotenoid production in inventive cells such that, as soon as a minimum desirable level of oleaginy is achieved, substantially all further carbon which is capable of being utilized and diverted into biosynthesis of products is diverted into a carotenoid production pathway. In some embodiments of the invention, this strategy involves engineering cells to be oleaginous; in other embodiments, it involves engineering cells to accumulate a higher level of lipid, particularly cytoplasmic lipid, than they would accumulate in the absence of such engineering even though the engineered cells may not become “oleaginous” as defined herein. In other embodiments, the extent to which an oleaginous host cell accumulates lipid is actually reduced so that remaining carbon can be utilized in carotenoid production.

Host Cells

Those of ordinary skill in the art will readily appreciate that a variety of yeast and fungal strains exist that are naturally oleaginous or that naturally produce carotenoids. Any of such strains may be utilized as host strains according to the present invention, and may be engineered or otherwise manipulated to generate inventive oleaginous, carotenoid-producing strains. Alternatively, strains that naturally are neither oleaginous nor carotenoid-producing may be employed. Furthermore, even when a particular strain has a natural capacity for oleaginy or for carotenoid production, its natural capabilities may be adjusted as described herein, so as to change the production level of lipid and/or carotenoid. In certain embodiments engineering or manipulation of a strain results in modification of a type of lipid and/or carotenoid which is produced. For example, a strain may be naturally oleaginous and/or carotenogenic, however engineering or modification of the strain may be employed so as to change the type of lipid which is accumulated and or to change the type of carotenoid which is produced.

When selecting a particular yeast or fungal strain for use in accordance with the present invention, it will generally be desirable to select one whose cultivation characteristics are amenable to commercial scale production. For example, it will generally (though not necessarily always) be desirable to avoid filamentous organisms, or organisms with particularly unusual or stringent requirements for growth conditions. However, where conditions for commercial scale production can be applied which allow for utilization of filamentous organisms, these may be selected as host cells. In some embodiments of the invention, it will be desirable to utilize edible organisms as host cells, as they may optionally be formulated directly into food or feed additives, or into nutritional supplements, as desired. For ease of production, some embodiments of the invention utilize host cells that are genetically tractable, amenable to molecular genetics (e.g., can be efficiently transformed, especially with established or available vectors; optionally can incorporate and/or integrate multiple genes, for example sequentially; and/or have known genetic sequence; etc), devoid of complex growth requirements (e.g., a necessity for light), mesophilic (e.g., prefer growth temperatures with in the range of about 25-32° C.), able to assimilate a variety of carbon and nitrogen sources and/or capable of growing to high cell density. Alternatively or additionally, various embodiments of the invention utilize host cells that grow as single cells rather than multicellular organisms (e.g., as mycelia).

In general, when it is desirable to utilize a naturally oleaginous organism in accordance with the present invention, any modifiable and cultivatable oleaginous organism may be employed. In certain embodiments of the invention, yeast or fungi of genera including, but not limited to, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, and Yarrowia are employed. In certain particular embodiments, organisms of species that include, but are not limited to, Blakeslea trispora, Candida pulcherrima, C. revkaufi, C. tropicalis, Cryptococcus curvatus, Cunninghamella echinulata, C. elegans, C. japonica, Lipomyces starkeyi, L. lipoferus, Mortierella alpina, M. isabellina, M. ramanniana, M. vinacea, Mucor circinelloides, Phycomyces blakesleanus, Pythium irregulare, Rhodosporidium toruloides, Rhodotorula glutinis, R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, Trichosporon pullans, T. cutaneum, and Yarrowia lipolytica are used.

Of these naturally oleaginous strains, some also naturally produce carotenoids and some do not. In most cases, only low levels (less than about 0.05% dry cell weight) of carotenoids are produced by naturally-occurring carotenogenic, oleaginous yeast or fungi. Higher levels of β-carotene are sometimes produced, but high levels of other carotenoids are generally not observed.

In general, any organism that is naturally oleaginous and non-carotenoid-producing (e.g., produce less than about 0.05% dry cell weight, do not produce the carotenoid of interest) may be utilized as a host cell in accordance with the present invention. In some embodiments, the organism is a yeast or fungus from a genus such as, but not limited to, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Pythium, Trichosporon, and Yarrowia; in some embodiments, the organism is of a species including, but not limited to, Mortierella alpina and Yarrowia lipolytica.

Comparably, the present invention may utilize any naturally oleaginous, carotenoid-producing organism as a host cell. In general, the present invention may be utilized to increase carbon flow into the isoprenoid pathway in naturally carotenoid-producing organisms (particularly for organisms other than Blakeslea and Phycomyces), and/or to shift production from one carotenoid (e.g., β-carotene) to another (e.g., astaxanthin). Introduction of one or more carotenogenic modifications (e.g., increased expression of one or more endogenous or heterologous carotenogenic polypeptides), in accordance with the present invention, can achieve these goals.

In certain embodiments of the invention, the utilized oleaginous, carotenoid-producing organism is a yeast or fungus, for example of a genus such as, but not limited to, Blakeslea, Mucor, Phycomyces, Rhodosporidium, and Rhodotorula; in some embodiments, the organism is of a species such as, Mucor circinelloides and Rhodotorula glutinis.

When it is desirable to utilize strains that are naturally non-oleaginous as host cells in accordance with the present invention, genera of non-oleaginous yeast or fungi include, but are not limited to, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); in some embodiments, the organism is of a species including, but not limited to, Aspergillus nidulans, A. niger, A. terreus, Botrytis cinerea, Cercospora nicotianae, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, K. lactis, Neurospora crassa, Pichia pastoris, Puccinia distincta, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, and Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

It will be appreciated that the term “non-oleaginous”, as used herein, encompasses both strains that naturally have some ability to accumulate lipid, especially cytoplasmically, but do not do so to a level sufficient to qualify as “oleaginous” as defined herein, as well as strains that do not naturally have any ability to accumulate extra lipid, e.g., extra-membranous lipid. It will further be appreciated that, in some embodiments of the invention, it will be sufficient to increase the natural level of oleaginy of a particular host cell, even if the modified cell does not qualify as oleaginous as defined herein.

As with the naturally oleaginous organisms, some of the naturally non-oleaginous fungi naturally produce carotenoids, whereas others do not. Genera of naturally non-oleaginous fungi that do not naturally produce carotenoids (e.g., produce less than about 0.05% dry cell weight, do not produce carotenoid of interest) may desirably be used as host cells in accordance with the present invention include, but are not limited to, Aspergillus, Kluyveromyces, Penicillium, Saccharomyces, and Pichia; species include, but are not limited to, Aspergillus niger and Saccharomyces cerevisiae. Genera of naturally non-oleaginous fungi that do naturally produce carotenoids and that may desirably be used as host cells in accordance with the present invention include, but are not limited to, Botrytis, Cercospora, Fusarium (Gibberella), Neurospora, Puccinia, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); species include, but are not limited to, Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

As discussed above, any of a variety of organisms may be employed as host cells in accordance with the present invention. In certain embodiments of the invention, host cells will be Yarrowia lipolytica cells. Advantages of Y. lipolytica include, for example, tractable genetics and molecular biology, availability of genomic sequence (see, for example. Sherman et al. Nucleic Acids Res. 32(Database issue):D315-8, 2004), suitability to various cost-effective growth conditions, and ability to grow to high cell density. In addition, Y. lipolytica is naturally oleaginous, such that fewer manipulations may be required to generate an oleaginous, carotenoid-producing Y. lipolytica strain than might be required for other organisms. Furthermore, there is already extensive commercial experience with Y. lipolytica.

Saccharomyces cerevisiae is also a useful host cell in accordance with the present invention, particularly due to its experimental tractability and the extensive experience that researchers have accumulated with the organism. Although cultivation of Saccharomyces under high carbon conditions may result in increased ethanol production, this can generally be managed by process and/or genetic alterations.

Additional useful hosts include Xanthophyllomyces dendrorhous (Phaffia rhodozyma), which is experimentally tractable and naturally carotenogenic. Xanthophyllomyces dendrorhous (Phaffia rhodozyma) strains can produce several carotenoids, including astaxanthin.

Aspergillus niger and Mortierella alpina accumulate large amounts of citric acid and fatty acid, respectively; Mortierella alpina is also oleaginous.

Neurospora or Gibberella are also useful. They are not naturally oleaginous and tend to produce very low levels of carotenoids, thus extensive modification may be required in accordance with the present invention. Neurospora and Gibberella are considered relatively tractable from an experimental standpoint. Both are filamentous fungi, such that production at commercial scales can be a challenge necessary to overcome in utilization of such strains.

Mucor circinelloides is another available useful species. While its molecular genetics are generally less accessible than are those of some other organisms, it naturally produces β-carotene, thus may require less modification than other species available.

Molecular genetics can be performed in Blakeslea, though significant effort may be required. Furthermore, cost-effective fermentation conditions can be challenging, as, for example, it may be required that the two mating types are mixed. Fungi of the genus Phycomyces are also possible sources which have the potential to pose fermentation process challenges, and these fungi are also may be less amenable to manipulate than several other potential host organisms.

Those of ordinary skill in the art will appreciate that the selection of a particular host cell for use in accordance with the present invention will also affect, for example, the selection of expression sequences utilized with any heterologous polypeptide to be introduced into the cell, and will also influence various aspects of culture conditions, etc. Much is known about the different gene regulatory requirements, protein targeting sequence requirements, and cultivation requirements, of different host cells to be utilized in accordance with the present invention (see, for example, with respect to Yarrowia, Barth et al. FEMS Microbiol Rev. 19:219, 1997; Madzak et al. J Biotechnol. 109:63, 2004; see, for example, with respect to Xanthophyllomyces, Verdoes et al. Appl Environ Microbiol 69: 3728-38, 2003; Visser et al. FEMS Yeast Res 4: 221-31, 2003; Martinez et al. Antonie Van Leeuwenhoek. 73(2):147-53, 1998; Kim et al. Appl Environ Microbiol. 64(5):1947-9, 1998; Wery et al. Gene. 184(1):89-97, 1997; see, for example, with respect to Saccharomyces, Guthrie and Fink Methods in Enzymology 194:1-933, 1991). In certain aspects, for example, targeting sequences of the host cell (or closely related analogs) may be useful to include for directing heterologous proteins to subcellular localization. Thus, such useful targeting sequences can be added to heterologous sequence for proper intracellular localization of activity. In other aspects (e.g., addition of mitochondrial targeting sequences), heterologous targeting sequences may be eliminated or altered in the selected heterologous sequence (e.g., alteration or removal of source organism plant chloroplast targeting sequences).

Engineering Oleaginy

All living organisms synthesize lipids for use in their membranes and various other structures. However, most organisms do not accumulate in excess of about 10% of their dry cell weight as total lipid, and most of this lipid generally resides within cellular membranes.

Significant biochemical work has been done to define the metabolic enzymes necessary to confer oleaginy on microorganisms (primarily for the purpose of engineering single cell oils as commercial sources of arachidonic acid and docosahexaenoic acid; see for example Ratledge Biochimie 86:807, 2004, the entire contents of which are incorporated herein by reference). Although this biochemical work is compelling, prior to the present invention, there have been no reports of de novo oleaginy being established through genetic engineering with the genes encoding the key metabolic enzymes.

It should be noted that oleaginous organisms typically only accumulate lipid when grown under conditions of carbon excess and nitrogen or other nutrient limitation. Under these conditions, the organism readily depletes the limiting nutrient but continues to assimilate the carbon source. The “excess” carbon is channeled into lipid biosynthesis so that lipids (usually triacylglycerols) accumulate in the cytosol, typically in the form of bodies.

In general, it is thought that, in order to be oleaginous, an organism must produce both acetyl-CoA and NADPH in the cytosol, which can then be utilized by the fatty acid synthase machinery to generate lipids. In at least some oleaginous organisms, acetyl-CoA is generated in the cytosol through the action of ATP-citrate lyase, which catalyzes the reaction:

citrate+CoA+ATP→acetyl-CoA+oxaloacetate+ADP+P_(i).  (1)

Of course, in order for ATP-citrate lyase to generate appropriate levels of acetyl-CoA in the cytosol, it must first have an available pool of its substrate citric acid. Citric acid is generated in the mitochondria of all eukaryotic cells through the tricarboxylic acid (TCA) cycle, and can be moved into the cytosol (in exchange for malate) by citrate/malate translocase.

In most oleaginous organisms, and in some non-oleaginous organisms, the enzyme isocitrate dehydrogenase, which operates as part of the TCA cycle in the mitochondria, is strongly AMP-dependent. Thus, when AMP is depleted from the mitochondria, this enzyme is inactivated. When isocitrate dehydrogenase is inactive, isocitrate accumulates in the mitochondria. This accumulated isocitrate is then equilibrated with citric acid, presumably through the action of aconitase. Therefore, under conditions of low AMP, citrate accumulates in the mitochondria. As noted above, mitochondrial citrate is readily transported into the cytosol.

AMP depletion, which in oleaginous organisms is believed to initiate the cascade leading to accumulation of citrate (and therefore acetyl-CoA) in the cytoplasm, occurs as a result of the nutrient depletion mentioned above. When oleaginous cells are grown in the presence of excess carbon source but under conditions limiting for nitrogen or some other nutrient(s), the activity of AMP deaminase, which catalyzes the reaction:

AMP→inosine 5′-monophosphate+NH₃  (2)

is strongly induced. The increased activity of this enzyme depletes cellular AMP in both the cytosol and the mitochondria. Depletion of AMP from the mitochondria is thought to inactivate the AMP-dependent isocitrate dehydrogenase, resulting in accumulation of citrate in the mitochondria and, therefore, the cytosol. This series of events is depicted diagrammatically in FIG. 2.

As noted above, oleaginy requires both cytosolic acetyl-CoA and cytosolic NADPH. It is believed that, in many oleaginous organisms, appropriate levels of cytosolic NADPH are provided through the action of malic enzyme (Enzyme 3 in FIG. 2). Some oleaginous organisms (e.g., Lipomyces and some Candida) do not appear to have malic enzymes, however, so apparently other enzymes can provide comparable activity, although it is expected that a dedicated source of NADPH is probably required for fatty acid synthesis (see, for example, Wynn et al., Microbiol 145:1911, 1999; Ratledge Adv. Appi. Microbiol. 51:1, 2002, each of which is incorporated herein by reference in its entirety).

Thus, according to the present invention, the oleaginy of a host organism may be enhanced by modifying the expression or activity of one or more polypeptides involved in generating cytosolic acetyl-CoA and/or NADPH. For example, modification of the expression or activity of one or more of acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminase can enhance oleaginy in accordance with the present invention. Exemplary polypeptides which can be utilized or derived so as to enhance oleaginy in accordance with the present invention include, but are not limited to those acetyl-CoA carboxylase, pyruvate decarboxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminase polypeptides provided in Table 1, Table 2, Table 3, Table 4, Table 5, and Table 6, respectively.

In some embodiments of the invention, where an oleaginous host cell is employed, enzymes and regulatory components relevant to oleaginy are already in place but could be modified, if desired, by for example altering expression or activity of one or more oleaginic polypeptides and/or by introducing one or more heterologous oleaginic polypeptides. In those embodiments of the invention where a non-oleaginous host cell is employed, it is generally expected that at least one or more heterologous oleaginic polypeptides will be introduced.

The present invention contemplates not only introduction of heterologous oleaginous polypeptides, but also adjustment of expression or activity levels of heterologous or endogenous oleaginic polypeptides, including, for example, alteration of constitutive or inducible expression patterns. In some embodiments of the invention, expression patterns are adjusted such that growth in nutrient-limiting conditions is not required to induce oleaginy. For example, genetic modifications comprising alteration and/or addition of regulatory sequences (e.g., promoter elements, terminator elements) may be utilized to confer particular regulation of expression patterns. Such genetic modifications may be utilized in conjunction with endogenous genes (e.g., for regulation of endogenous oleagenic polypeptide(s)); alternatively, such genetic modifications may be included so as to confer regulation of expression of at least one heterologous polypeptide (e.g., oleagenic polypeptide(s)). For example, promoters including, but not limited to Tef1, Gpd1 promoters can be used in conjunction with endogenous genes and/or heterolous genes for modification of expression patterns of endogenous oleaginic polypeptides and/or heterolous oleagenic polypeptides. Similarly, exemplary terminator sequences include, but are not limited to, use of Y. lipolytica XPR2 terminator sequences.

In some embodiments, at least one oleaginic polypeptide is introduced into a host cell. In some embodiments of the invention, a plurality (e.g., two or more) of different oleaginic polypeptides is introduced into the same host cell. In some embodiments, the plurality of oleaginic polypeptides contains polypeptides from the same source organism; in other embodiments, the plurality includes polypeptides independently selected from different source organisms.

Representative examples of a variety of oleaginic polypeptides that may be introduced into or modified within host cells according to the present invention, include, but are not limited to, those provided in Tables 1-6. As noted above, it is expected that at least some of these polypeptides (e.g., malic enzyme and ATP-citrate lyase) should desirably act in concert, and possibly together with one or more components of fatty acid synthase, such that, in some embodiments of the invention, it will be desirable to utilize two or more oleaginic polypeptides from the same source organism.

In general, source organisms for oleaginic polypeptides to be used in accordance with the present invention include, but are not limited to, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia). In some embodiments, the source species for acetyl CoA carboxylase, ATP-citrate lyase, malice enzyme and/or AMP deaminase polypeptides include, but are not limited to, Aspergillus nidulans, Cryptococcus neoformans, Fusarium fujikuroi, Kluyveromyces lactis, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, and Yarrowia lipolytica; in some embodiments, source species for pyruvate decarboxylase or isocitrate dehydrogenase polypeptides include, but are not limited to Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Rhodotorula glutinis, Candida utilis, Mortierella alpina and Yarrowia lipolytica.

Engineering Carotenoid Production

Carotenoids are synthesized from isoprenoid precursors, some of which are also involved in the production of steroids and sterols. The most common isoprenoid biosynthesis pathway, sometimes referred to as the “mevalonate pathway”, is generally depicted in FIG. 3. As shown, acetyl-CoA is converted, via hydroxymethylglutaryl-CoA (HMG-CoA), into mevalonate. Mevalonate is then phosphorylated and converted into the five-carbon compound isopentenyl pyrophosphate (IPP). Following isomerization of IPP into dimethylallyl pyrophosphate (DMAPP), three sequential condensation reactions with additional molecules of IPP generate the ten-carbon molecule geranyl pyrophosphate (GPP), followed by the fifteen-carbon molecule farnesyl pyrophosphate (FPP), and finally the twenty-carbon compound geranylgeranyl pyrophosphate (GGPP).

An alternative isoprenoid biosynthesis pathway, that is utilized by some organisms (particularly bacteria) and is sometimes called the “mevalonate-independent pathway”, is depicted in FIG. 4. This pathway is initiated by the synthesis of 1-deoxy-D-xyloglucose-5-phosphate (DOXP) from pyruvate and glyceraldehyde-3-phosphate. DOXP is then converted, via a series of reactions shown in FIG. 4, into IPP, which isomerizes into DMAPP and is then converted, via GPP and FPP, into GGPP as shown in FIG. 3 and discussed above.

Various proteins involved in isoprenoid biosynthesis have been identified and characterized in a number of organisms. Moreover, various aspects of the isoprenoid biosynthesis pathway are conserved throughout the fungal, bacterial, plant and animal kingdoms. For example, polypeptides corresponding to the acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, IPP isomerase, FPP synthase, and GGPP synthase shown in FIG. 3 have been identified in and isolated from a wide variety of organisms and cells. Representative examples of a wide variety of such polypeptides are provided in Tables 7-15. One or more of the polypeptides selected from those provided in any one of Tables 7-15 may be utilized or derived for use in the methods and compositions in accordance with the present invention.

According to the present invention, carotenoid production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in isoprenoid biosynthesis. In some embodiments, such modification involves introduction of one or more heterologous isoprenoid biosynthesis polypeptides into the host cell; alternatively or additionally, modifications may be made to the expression or activity of one or more endogenous or heterologous isoprenoid biosynthesis polypeptides. Given the considerable conservation of components of the isoprenoid biosynthesis polypeptides, it is expected that heterologous isoprenoid biosynthesis polypeptides will often function even in significantly divergent organisms. Furthermore, should it be desirable to introduce more than one heterologous isoprenoid biosynthesis polypeptide, in many cases polypeptides from different source organisms will function together. In some embodiments of the invention, a plurality of different heterologous isoprenoid biosynthesis polypeptides is introduced into the same host cell. In some embodiments, this plurality contains only polypeptides from the same source organism (e.g., two or more sequences of, or sequences derived from, the same source organism); in other embodiments the plurality includes polypeptides independently selected from different source organisms (e.g., two or more sequences of, or sequences derived from, at least two independent source organisms).

In some embodiments of the present invention that utilize heterologous isoprenoid biosynthesis polypeptides, the source organisms include, but are not limited to, fungi of the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Schizosaccharomyces, Sclerotium, Trichoderms, Ustilago, and Xanthophyllomyces (Phaffia). In certain embodiments, the source organisms are of a species including, but not limited to, Cryptococcus neoformans, Fusarium fujikuroi, Kluyverimyces lactis, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, and Yarrowia lipolytica.

As noted above, the isoprenoid biosynthesis pathway is also involved in the production of non-carotenoid compounds, such as sterols, steroids, and vitamins, such as vitamin E or vitamin K. Proteins that act on isoprenoid biosynthesis pathway intermediates, and divert them into biosynthesis of non-carotenoid compounds are therefore indirect inhibitors of carotenoid biosynthesis (see, for example, FIG. 5, which illustrates points at which isoprenoid intermediates are channeled into other biosynthesis pathways). Such proteins are therefore considered isoprenoid biosynthesis competitor polypeptides. Reductions of the level or activity of such isoprenoid biosynthesis competitor polypeptides are expected to increase carotenoid production in host cells according to the present invention.

In some embodiments of the present invention, production or activity of endogenous isoprenoid biosynthesis competitor polypeptides may be reduced or eliminated in host cells. In some embodiments, this reduction or elimination of the activity of an isoprenoid biosynthesis competitor polypeptide can be achieved by treatment of the host organism with small molecule inhibitors of enzymes of the ergosterol biosynthetic pathway. Enzymes of the ergosterol biosynthetic pathway include, for example, squalene synthase, squalene epoxidase, 2,3-oxidosqualene-lanosterol cyclase, cytochrome P450 lanosterol 14α-demethylase, C-14 sterol reductase, C-4 sterol methyl oxidase, SAM:C-24 sterol methyltransferase, C-8 sterol isomerase, C-5 sterol desaturase, C-22 sterol desaturase, and C-24 sterol reductase. Each of these enzymes is considered an isoprenoid biosynthesis competitor polypeptide. Regulators of these enzymes may also be considered isoprenoid biosynthesis competitor polypeptides (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 GI:2133159) and Mot3 (Genbank Accession NP_(—)013786 GI:6323715), which may or may not have homologs in other organisms.

In other embodiments, reduction or elimination of the activity of an isoprenoid biosynthesis competitor polypeptide can be achieved by decreasing activity of the ubiquinone biosynthetic pathway. The commitment step in ubiquinone biosynthesis is the formation of para-hydroxybenzoate (PHB) from tyrosine or phenylalanine in mammals or chorismate in bacteria, followed by condensation of PHB and isoprene precursor, resulting in addition of the prenyl group. This reaction is catalyzed by PHB-polyprenyltransferase. The isoprenoid side chain of ubiquinone is determined by the prenyldiphosphate synthase enzyme. The 3-decaprenyl-4-hydroxybenzoic acid resulting from the condensation of PHB and decaprenyldiphosphate reaction undergoes further modifications, which include hydroxylation, methylation and decarboxylation, in order to form ubiquinone (CoQ10). Thus, inhibition of prenyldiphosphate synthase leading from farnesyldiphosphate to extended isoprenoids, or inhibition of PHB polyprenyltransferase may be useful in increasing the amount of isoprenoid available for carotenoid biosynthesis. (Examples of prenyldiphosphate synthase and PHB-polyprenyltransferase enzymes are depicted in Tables 29 and 30, respectively).

Known small molecule inhibitors of isoprenoid biosynthesis competitor enzymes include, but are not limited to, zaragosic acid (including analogs thereof such as TAN1607A (Biochem Biophys Res Commun 1996 Feb. 15; 219(2):515-520)), RPR 107393 (3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo[2-2-2]octane dihydrochloride; J Pharmacol Exp Ther. 1997 May; 281(2):746-52), ER-28448 (5-{N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino}-1,1-penthylidenebis(phosphonic acid) trisodium salt; Journal of Lipid Research, Vol. 41, 1136-1144, July 2000), BMS-188494 (The Journal of Clinical Pharmacology, 1998; 38:1116-1121), TAK-475 (1-[2-[(3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl]acetyl]piperidin-4-acetic acid; Eur J. Pharmacol. 2003 Apr. 11; 466(1-2):155-61), YM-53601 ((E)-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride; Br J. Pharmacol. 2000 September; 131(1):63-70), or squalestatin I that inhibit squalene synthase; terbinafine that inhibits squalene epoxidase; various azoles that inhibit cytochrome P450 lanosterol 14α-demethylase; and fenpropimorph that inhibits the C-14 sterol reductase and the C-8 sterol isomerase. In other embodiments, heterologous isoprenoid biosynthesis competitor polypeptides may be utilized (whether functional or non-functional; in some embodiments, dominant negative mutants are employed).

One particular isoprenoid biosynthesis competitor polypeptide useful according to the present invention is squalene synthase which has been identified and characterized from a variety of organisms; representative examples of squalene synthase polypeptide sequences are included in Table 16. In some embodiments of the invention that utilize squalene synthase (or modifications of squalene synthase) source organisms include, but are not limited to, Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Aspergillus niger, Saccharomyces cerevisiae, Mucor circinelloides, Rhotorula glutinis, Candida utilis, Mortierella alpina, and Yarrowia lipolytica.

The carotenoid biosynthesis pathway branches off from the isoprenoid biosynthesis pathway at the point where GGPP is formed. The commitment step in carotenoid biosynthesis is the formation of phytoene by the head-to-head condensation of two molecules of GGPP, catalyzed by phytoene synthase (often called crtB; see FIG. 6). A series of dehydrogenation reactions, each of which increases the number of conjugated double bonds by two, converts phytoene into lycopene via neurosporene. The pathway branches at various points, both before and after lycopene production, so that a wide range of carotenoids can be generated. For example, action of a cyclase enzyme on lycopene generates γ-carotene; action of a desaturase instead produces 3,4-didehydrolycopene. γ-carotene is converted to β-carotene through the action of a cyclase. β-carotene can be processed into any of a number of products (see, for example, FIG. 6C), including astaxanthin (via echinone, hydroxyechinone, and phoenicoxanthin).

According to the present invention, carotenoid production in a host organism may be adjusted by modifying the expression or activity of one or more proteins involved in carotenoid biosynthesis. As indicated, in some embodiments, it will be desirable to utilize as host cells organisms that naturally produce one or more carotenoids. In some such cases, the focus will be on increasing production of a naturally-produced carotenoid, for example by increasing the level and/or activity of one or more proteins involved in the synthesis of that carotenoid and/or by decreasing the level or activity of one or more proteins involved in a competing biosynthetic pathway. Alternatively or additionally, in some embodiments it will be desirable to generate production of one or more carotenoids not naturally produced by the host cell.

According to some embodiments of the invention, it will be desirable to introduce one or more heterologous carotenogenic polypeptides into a host cell. As will be apparent to those of ordinary skill in the art, any of a variety of heterologous polypeptides may be employed; selection will consider, for instance, the particular carotenoid whose production is to be enhanced. The present invention contemplates not only introduction of heterologous carotenogenic polypeptides, but also adjustment of expression or activity levels of heterologous or endogenous carotenogenic polypeptides, including, for example, alteration of constitutive or inducible expression patterns. In some embodiments of the invention, expression patterns are adjusted such that growth in nutrient-limiting conditions is not required to induce oleaginy. For example, genetic modifications comprising alteration and/or addition of regulatory sequences (e.g., promoter elements, terminator elements) may be utilized to confer particular regulation of expression patterns. Such genetic modifications may be utilized in conjunction with endogenous genes (e.g., for regulation of endogenous carotenogenic); alternatively, such genetic modifications may be included so as to confer regulation of expression of at least one heterologous polypeptide (e.g., carotenogenic polypeptide(s)). For example, promoters including, but not limited to Tef1, Gpd1 promoters can be used in conjunction with endogenous genes and/or heterolous genes for modification of expression patterns of endogenous carotenogenic polypeptide(s) and/or heterolous carotenogenic polypeptide(s). Similarly, exemplary terminator sequences include, but are not limited to, use of Y. lipolytica XPR2 terminator sequences.

As indicated in FIG. 6 and in the literature, proteins involved in carotenoid biosynthesis include, but are not limited to, phytoene synthase, phytoene dehydrogenase, lycopene cyclase, carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase (a single multifunctional enzyme found in some source organisms that typically has both ketolase and hydroxylase activities), carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilon subunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerol acyltransferase. Representative example sequences for these carotenoid biosynthesis polypeptides are provided in Tables 17-25.

Xanthophylls can be distinguished from other carotenoids by the presence of oxygen containing functional groups on their cyclic end groups. For instance, lutein and zeaxanthin contain a single hydroxyl group on each of their terminal ring structures, while astaxanthin contains both a keto group and a hydroxyl on each terminal ring. This property makes xanthophylls more polar than carotenes such as beta-carotene and lycopene, and thus dramatically reduces their solubility in fats and lipids. Naturally occurring xanthophylls are often found as esters of the terminal hydroxyl groups, both mono- and diesters of fatty acids. They also occur as glucosides in certain species of bacteria. The solubility and dispersibility of xanthophylls can be greatly modified by the addition of ester moieties, and it is known that esterification can also affect the absorbability and/or bioavailability of a given carotenoid. It is an objective of this invention to maximize the amount of a particular xanthophyll accumulating within the intracellular triacylglyceride fraction of oleaginous yeasts, and one mechanism for achieving this goal is to increase the hydrophobic nature of the xanthophyll product that accumulates. One way of achieving this is to engineer the production of fatty-acyl mono- and/or diesters of the target xanthophyll compound.

A variety of enzymes can function to esterify carotenoids. For example, carotenoid glucosyltransferases have been identified in several bacterial species (see, e.g., Table 24). In addition, acyl CoA:diacyglycerol acyltransferase (DGAT) and acyl CoA:monoacylglycerol acyltransferases (MGAT), which function in the final steps of triacylglycerol biosynthesis, are likely to serve an additional role in the esterification of xanthophylls. Representative DGAT polypetides are shown in Table 25. Furthermore, other enzymes may specifically modify carotenoids and molecules of similar structure (e.g. sterols) and be available for modification and ester production.

In some embodiments of the invention, potential source organisms for carotenoid biosynthesis polypeptides include, but are not limited to, genera of naturally oleaginous or non-oleaginous fungi that naturally produce carotenoids. These include, but are not limited to, Botrytis, Cercospora, Fusarium (Gibberella), Mucor, Neurospora, Phycomyces, Puccina, Rhodotorula, Sclerotium, Trichoderma, and Xanthophyllomyces. Exemplary species include, but are not limited to, Neurospora crassa, Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Mucor circinelloides, and Rhodotorula glutinis. Of course, carotenoids are produced by a wide range of diverse organisms such as plants, algae, yeast, fungi, bacteria, cyanobacteria, etc. Any such organisms may be source organisms for carotenoid biosynthesis polypeptides according to the present invention.

It will be appreciated that the particular carotenogenic modification to be applied to a host cell in accordance with the present invention will be influenced by which carotenoid(s) is desired to be produced. For example, isoprenoid biosynthesis polypeptides are relevant to the production of most carotenoids. Carotenoid biosynthesis polypeptides are also broadly relevant. Ketolase is particularly relevant for production of canthaxanthin, as hydroxylase is for production of lutein and zeaxanthin, among others. Both hydroxylase and ketolase (or astaxanthin synthase) are particularly useful for production of astaxanthin.

Production and Isolation of Carotenoids

As discussed above, accumulation of lipid bodies in oleaginous organisms is generally induced by growing the relevant organism in the presence of excess carbon source and limiting nitrogen. Specific conditions for inducing such accumulation have previously been established for a number of different oleaginous organisms (see, for example, Wolf (ed.) Nonconventional yeasts in biotechnology Vol. 1, Springer-Verlag, Berlin, Germany, pp. 313-338; Lipids 18(9):623, 1983; Indian J. Exp. Biol. 35(3):313, 1997; J. Ind. Microbiol. Biotechnol. 30(1):75, 2003; Bioresour Technol. 95(3):287, 2004, each of which is incorporated herein by reference in its entirety).

In general, it will be desirable to cultivate inventive modified host cells under conditions that allow accumulation of at least about 20% of their dry cell weight as lipid. In other embodiments, the inventive modified host cells are grown under conditions that permit accumulation of at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or even 80% or more of their dry cell weight as lipid. In certain embodiments, the host cells utilized are cells which are naturally oleaginous, and induced to produce lipid to the desired levels. In other embodiments, the host cells are cells which naturally produce lipid, but have been engineered to increase production of lipid such that desired levels of lipid production and accumulation are achieved.

In certain embodiments, the host cells of the invention are not naturally oleaginous, but have been engineered to produce lipid such that desired levels of lipid production are obtained. Those of ordinary skill in the art will appreciate that, in general, growth conditions that are effective for inducing lipid accumulation in a source organism, may well also be useful for inducing lipid accumulation in a host cell into which the source organism's oleaginic polypeptides have been introduced. Of course, modifications may be required in light of characteristics of the host cell, which modifications are within the skill of those of ordinary skill in the art.

It will also be appreciated by those of ordinary skill in the art that it will generally be desirable to ensure that production of the desired carotenoid by the inventive modified host cell occurs at an appropriate time in relation to the induction of oleaginy such that the carotenoid(s) accumulate(s) in the lipid bodies. In some embodiments, it will be desirable to induce production of the carotenoid(s) in a host cell which does not naturally produce the carotenoid(s), such that detectable levels of the carotenoid(s) is/are produced. In certain aspects the host cells which do not naturally produce a certain carotenoid(s) are capable of production of other carotenoid(s) (e.g. certain host cells may, for example, naturally produce β-carotene but may not naturally produce astaxanthin); in other aspects the host cells do not naturally produce any carotenoid(s). In other embodiments, it will be desirable to increase production levels of carotenoid(s) in a host cell which does naturally produce low levels of the carotenoid(s), such that increased detectable levels of the carotenoid(s) are produced. In certain aspects, the host cells which do naturally produce the carotenoid(s) (e.g., β-carotene) also produce additional carotenoid(s) (e.g., astaxanthin, etc.); in still other aspects, the cells which naturally produce the carotenoid(s) (e.g., β-carotene) do not produce additional carotenoid(s).

In certain embodiments of the invention, it will be desirable to accumulate carotenoids to levels (i.e., considering the total amount of all produced carotenoids together) that are greater than at least about 1% of the dry weight of the cells. In some embodiments, the total carotenoid accumulation in the lipid bodies will be to a level at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% or more of the total dry weight of the cells. In certain embodiments of the invention, it will be desirable to achieve total levels of carotenoid accumulation in the lipid bodies (i.e., considering the total amount of all produced carotenoids together) that are greater than at least about 1% of the dry weight of the cells. In some embodiments, the total carotenoid accumulation in the lipid bodies will be to a level at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% or more of the total dry weight of the cells.

Bacterial carotenogenic genes have already been demonstrated to be transferrable to other organisms, and are therefore particularly useful in accordance with the present invention (see, for example, Miura et al., Appl. Environ. Microbiol. 64:1226, 1998). In other embodiments, it may be desirable to utilize genes from other source organisms such as plant, alga, or microalgae; these organisms provide a variety of potential sources for ketolase and hydroxylase polypeptides. Still additional useful source organisms include fungal, yeast, insect, protozoal, and mammalian sources of polypeptides.

In certain embodiments, the Mucor circinelloides multi-functional phytoene synthase/lycopene cyclase and the Neurospora crassa phytoene dehydrogenase genes can be expressed in Yarrowia lipolytica. Subsequent overexpression of the catalytic domain from N. crassa hydroxymethylglutaryl-CoA reductase and/or treatment of the modified Y. lipolytica strains with the squalene synthase inhibitor zaragozic acid further increases carotenoid production. Finally, Paracoccus marcusii genes encoding carotenoid hydroxylase and carotenoid ketolase enzymes are expressed in Y. lipolytica β-carotene-producing strains, and this modification results in the accumulation of astaxanthin. Similar approaches to enhance carotenoid production could be employed in other oleaginous or non-oleaginous host organisms can be undertaken, using the same, homologous, or functionally similar carotogenic polypeptides.

It should be noted that, for inventive organisms that produce more than one carotenoid, it will sometimes be possible to adjust the relative amounts of individual carotenoids produced by adjusting growth conditions. For example, it has been reported that controlling the concentration of dissolved oxygen in a culture during cultivation can regulate relative production levels of certain carotenoids such as β-carotene, echinenone, β-cryptoxanthin, 3-hydroxyechinenone, asteroidenone, canthaxanthin, zeaxanthin, adonirubin, adonixanthin and astaxanthin (see, for example, U.S. Pat. No. 6,825,002 to Tsubokura et al., the entire contents of which are incorporated herein by reference).

Particularly for embodiments of the present invention directed toward production of astaxanthin, it will often be desirable to utilize one or more genes from a natural astaxanthin-producing organism. Where multiple heterologous polypeptides are to be expressed, it may be desirable to utilize the same source organism for all, or to utilize closely related source organisms.

One advantage provided by the present invention is that, in addition to allowing the production of high levels of carotenoids, the present invention allows those produced compounds to be readily isolated because they accumulate in the lipid bodies within oleaginous organisms. Methods and systems for isolating lipid bodies have been established for a wide variety of oleaginous organisms (see, for example, U.S. Pat. Nos. 5,164,308; 5,374,657; 5,422,247; 5,550,156; 5,583,019; 6,166,231; 6,541,049; 6,727,373; 6,750,048; and 6,812,001, each of which is incorporated herein by reference in its entirety). In brief, cells are typically recovered from culture, often by spray drying, filtering or centrifugation. In some instances, cells are homogenized and then subjected to supercritical liquid extraction or solvent extraction (e.g., with solvents such as chloroform, hexane, methylene chloride, methanol, isopropanol, ethyl acetate, etc.), yielding a crude oil suspension. This oil suspension may optionally be refined as known in the art. Refined oils may be used directly as feed or food additives. Alternatively or additionally, carotenoids can be isolated from the oil using conventional techniques.

Given the sensitivity of carotenoids generally to oxidation, many embodiments of the invention employ oxidative stabilizers (e.g., tocopherols, vitamin C; ethoxyquin; vitamin E, BHT, BHA, TBHQ, etc, or combinations thereof) during and/or after carotenoid isolation. Alternatively or additionally, microencapsulation, for example with proteins, may be employed to add a physical barrier to oxidation and/or to improve handling (see, for example, U.S. Patent Application 2004/0191365).

Uses

Carotenoids produced according to the present invention can be utilized in any of a variety of applications, for example exploiting their biological or nutritional properties (e.g., anti-oxidant, anti-proliferative, etc.) and/or their pigment properties. For example, according to the present invention, carotenoids may be used in pharmaceuticals (see, for example, Bertram, Nutr. Rev. 57:182, 1999; Singh et al., Oncology 12:1643, 1998; Rock, Pharmacol. Ther. 75:185, 1997; Edge et al, J. Photochem Photobiol 41:189, 1997; U.S. Patent Application 2004/0116514; U.S. Patent Application 2004/0259959), food supplements (see, for example, Koyama et al, J. Photochem Photobiol 9:265, 1991; Bauernfeind, Carotenoids as colorants and vitamin A precursors, Academic Press, NY, 1981; U.S. Patent Application 2004/0115309; U.S. Patent Application 2004/0234579), electro-optic applications, animal feed additives (see, for example, Krinski, Pure Appl. Chem. 66:1003, 1994; Polazza et al., Meth. Enzymol. 213:403, 1992), cosmetics (as anti-oxidants and/or as cosmetics, including fragrances; see for example U.S. Patent Application 2004/0127554), etc. Carotenoids produced in accordance with the present invention may also be used as intermediates in the production of other compounds (e.g., steroids, etc.).

For example, astaxanthin and/or esters thereof may be useful in a variety of pharmaceutical applications and health foods including treatment of inflammatory diseases, asthma, atopic dermatitis, allergies, multiple myeloma, arteriosclerosis, cardiovascular disease, liver disease, cerebrovascular disease, thrombosis, neoangiogenesis-related diseases, including cancer, rheumatism, diabetic retinopathy; macular degeneration and brain disorder, hyperlipidemia, kidney ischemia, diabetes, hypertension, tumor proliferation and metastasis; and metabolic disorders. Additionally, carotenoids and astaxanthin may be useful in the prevention and treatment of fatigue, for improving kidney function in nephropathy from inflammatory diseases, as well as prevention and treatment of other life habit-related diseases. Still further, astaxanthin has been found to play a role as inhibitors of various biological processes, including interleukin inhibitors, phosphodiesterase inhibitors inhibitors, phospholipase A2 inhibitors, cyclooxygenase-2 inhibitors, matrix metalloproteinase inhibitors, capillary endothelium cell proliferation inhibitors, lipoxygenase inhibitors. See, e.g., Japanese Publication No. 2006022121, published 20060126 (JP Appl No. 2005-301156 filed 20051017); Japanese Publication No. 2006016408, published 20060119 (JP Appl No. 2005-301155 filed 20051017); Japanese Publication No. 2006016409, published 20060119 (JP Appl No. 2005-301157 filed 20051017); Japanese Publication No. 2006016407, published 20060119 (JP Appl No. 2005-301153 filed 20051017); Japanese Publication No. 2006008717, published 20060112 (JP Appl No. 2005-301151 filed 20051017); Japanese Publication No. 2006008716, published 20060112 (JP Appl No. 2005-301150 filed 20051017); Japanese Publication No. 2006008720, published 20060112 (JP Appl No. 2005-301158 filed 20051017); Japanese Publication No. 2006008719, published 20060112 (JP Appl No. 2005-301154 filed 20051017); Japanese Publication No. 2006008718, published 20060112 (JP Appl No. 2005-301152 filed 20051017); Japanese Publication No. 2006008713, published 20060112 (JP Appl No. 2005-301147 filed 20051017); Japanese Publication No. 2006008715, published 20060112 (JP Appl No. 2005-301149 filed 20051017); Japanese Publication No. 2006008714, published 20060112 (JP Appl No. 2005-301148 filed 20051017); and Japanese Publication No. 2006008712, published 20060112 (JP Appl No. 2005-301146 filed 20051017).

It will be appreciated that, in some embodiments of the invention, carotenoids produced by manipulated host cells as described herein are incorporated into a final product (e.g., food or feed supplement, pharmaceutical, cosmetic, dye-containing item, etc.) in the context of the host cell. For example, host cells may be lyophilized, freeze dried, frozen or otherwise inactivated, and then whole cells may be incorporated into or used as the final product. The host cell may also be processed prior to incorporation in the product to increase bioavailability (e.g., via lysis). Alternatively or additionally, a final product may incorporate only a portion of the host cell (e.g., fractionated by size, solubility), separated from the whole. For example, in some embodiments of the invention, lipid droplets are isolated from the host cells and are incorporated into or used as the final product. In other embodiments, the carotenoids themselves, or individual carotenoid compounds are isolated and reformulated into the final product.

As stated above, fatty acid and glucoside esters are the predominant carotenoid esters found in nature, whereas additional esters (e.g. with organic acids or inorganic phosphate) can be synthesized to generate useful product forms. For delivery, carotenoid esters can also be formulated as salts of the ester form. See, e.g., US Publication No. 20050096477.

The amount of carotenoid incorporated into a given product may vary dramatically depending on the product, and the particular carotenoid(s) involved. Amounts may range, for example, from less than 0.01% by weight of the product, to more than 1%, 10%, 20%, 30% or more; in some cases the carotenoid may comprise 100% of the product.

In some embodiments of the invention, one or more produced carotenoids is incorporated into a component of food or feed (e.g., a food supplement). Types of food products into which carotenoids can be incorporated according to the present invention are not particularly limited, and include beverages such as teas, juices, and liquors; confections such as jellies and biscuits; fat-containing foods and beverages such as dairy products; processed food products such as rice and soft rice (or porridge); infant formulas; or the like. In some embodiments of this aspect of the invention, it may be useful to incorporate the carotenoids within bodies of edible lipids as it may facilitate incorporation into certain fat-containing food products.

Examples of feedstuffs into which carotenoids produced in accordance with the present invention may be incorporated include, for instance, pet foods such as cat foods, dog foods and the like, feeds for aquarium fish, cultured fish or crustaceans, etc., feed for farm-raised animals (including livestock and further including fish or crustaceans raised in aquaculture). Food or feed material into which the carotenoid(s) produced in accordance with the present invention is incorporated is preferably palatable to the organism which is the intended recipient. This food or feed material may have any physical properties currently known for a food material (e.g., solid, liquid, soft).

In some embodiments of the invention, one or more produced carotenoids is incorporated into a cosmetic product. Examples of such cosmetics include, for instance, skin cosmetics (e.g., lotions, emulsions, creams and the like), lipsticks, anti-sunburn cosmetics, makeup cosmetics, fragrances, products for daily use (e.g., toothpastes, mouthwashes, bad breath preventive agents, solid soaps, liquid soaps, shampoos, conditioners), etc.

In some embodiments, one or more produced carotenoids is incorporated into a pharmaceutical. Examples of such pharmaceuticals include, for instance, various types of tablets, capsules, drinkable agents, troches, gargles, etc. In some embodiments, the pharmaceutical is suitable for topical application. Dosage forms are not particularly limited, and include capsules, oils, granula, granula subtilae, pulveres, tabellae, pilulae, trochisci, or the like. Oils and oil-filled capsules may provide additional advantages both because of their lack of ingredient decomposition during manufacturing, and because inventive carotenoid-containing lipid droplets may be readily incorporated into oil-based formulations.

Pharmaceuticals according to the present invention may be prepared according to techniques established in the art including, for example, the common procedure as described in the United States Pharmacopoeia, for example.

Carotenoids produced according to the present invention may be incorporated into any pigment-containing product including, for example, fabric, paint, etc. They may also be incorporated into a product which is an environmental indicator, or an instrument such as a biosensor for use as a detection agent.

EXEMPLIFICATION

Table 26 below describes certain Yarrowia lipolytica strains used in the following exemplification:

TABLE 26 Yarrowia lipolytica strains. NRRL Y-1095 Wild type diploid ATCC76861 MATB ura2-21 lyc1-5 LYS1-5B ATCC76982 MATB ade1 leu2-35 lyc1-5 xpr2 ATCC201249 MATA ura3-302 leu2-270 lys8-11 PEX17-HA MF346 MATA ura2-21 ATCC76861 × ATCC201249 MF350 MATB ura2-21 leu2-35 ade1 ATCC76982 × MF346 (The genotypes at LYC1, LYS1, XPR2, and PEX17 were not determined in crosses nor verified for ATCC strains.)

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. or Ausubel et al. (Sambrook J, Fritsch E F, Maniatis T (eds). 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York; Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K (eds). 1998. Current Protocols in Molecular Biology. Wiley: New York).

Example 1 Production of Plasmids for Carotenoid Strain Construction

Plasmids were generated for construction of carotenoid producing strains. The following subparts describe production of plasmids encoding carotenogenic polypeptides. Plasmids used in these studies and details of their construction are described in Table 27. Additional plasmid construction details and descriptions of their use are found in the text of the relevant subsection. All PCR amplifications used NRRL Y-1095 genomic DNA as template unless otherwise specified. The URA5 gene described below is allelic with the ura1-21 auxotrophy above. The GPD1 and TEF1 promoters are from Y. lipolytica as is the XPR2 terminator.

GGS1 is the gene encoding the Y. lipolytica gene encoding geranylgeranylpyrophosphate synthase. The nucleic acid coding sequence, and encoded Ggs1 protein of pMB4591 and pMB4683 are as follows:

(SEQ ID NO: 1) atggattataacagcgcggatttcaaggagatatggggcaaggccgccgacaccgcgctgctgggaccgta caactacctcgccaacaaccggggccacaacatcagagaacacttgatcgcagcgttcggagcggttatca aggtggacaagagcgatctcgagaccatttcgcacatcaccaagattttgcataactcgtcgctgcttgtt gatgacgtggaagacaactcgatgctccgacgaggcctgccggcagcccattgtctgtttggagtccccca aaccatcaactccgccaactacatgtactttgtggctctgcaggaggtgctcaagctcaagtcttatgatg ccgtctccattttcaccgaggaaatgatcaacttgcatagaggtcagggtatggatctctactggagagaa acactcacttgccectcggaagacgagtatctggagatggtggtgcacaagaccggtggactgtttcggct ggctctgagacttatgctgtcggtggcatcgaaacaggaggaccatgaaaagatcaactttgatctcacac accttaccgacacactgggagtcatttaccagattctggatgattacctcaacctgcagtccacggaattg accgagaacaagggattctgcgaagatatcagcgaaggaaagttttcgtttccgctgattcacagcatacg caccaacccggataaccacgagattctcaacattctcaaacagcgaacaagcgacgcttcactcaaaaagt acgccgtggactacatgagaacagaaaccaagagtttcgactactgcctcaagaggatacaggccatgtca ctcaaggcaagttcgtacattgatgatctagcagcagctggccacgatgtctccaagctacgagccatttt gcattattttgtgtccacctctgactgtgaggagagaaagtactttgaggatgcgcagtga (SEQ ID NO: 2) mdynsadflceiwgkaadtallgpynylannrghnirehliaafgavikvdksdletishitkilhnssll vddvednsmlaglpaahclfgvpqtinsanymyfvalqevlklksydaysifteeminlhrgqgmdlywre tltcpsedeylemvvhktgglfflalrlmlsvaskqedhekinfdlthltdtlgviyqilddylnlqstel tenkgfcedisegkfsfplihsirtnpdnheilnilkgrtsdaslkkyavdymrtetksfdyclkriqams lkassyiddlaaaghdvsklrailhyfystsdceerkyfedaq.

TABLE 27 Plasmids Plasmid Backbone Insert Oligos or source pMB4529 PCR2.1 3.4 kb ADE1 PCR product MO4475 & MO4476 pMB4534 PCR2.1 2.1 kb LEU2 PCR product MO4477 & MO4478 pMB4535 PCR2.1 1.2 kb URA5 PCR product MO4471 & MO4472 pMB4589 pMB4535 (KpnI + SpeI) 1.2 kb GPD1 promoter MO4568 & (KpnI + NotI); 0.14 kb MO4591; MO4566 & XPR2 terminator MO4593 (NotI + SpeI) pMB4590 pMB4535 (KpnI + SpeI) 0.4 kb TEF1 promoter MO4571 & (KpnI + NotI); 0.14 kb MO4592; MO4566 & XPR2 terminator MO4593 (NotI + SpeI) pMB4591 pMB4592 (NheI + MluI) 1.0 kb GGS1 ORF MO4534 & (XbaI + MluI) MO4544 pMB4597 pMB4534 (Acc65I + SpeI) GPD1 promoter & XPR2 From pMB4589 terminator (Acc65I + SpeI) pMB4603 pMB4597 (RsrII + MluI) Residual backbone & TEF1 From pMB4590 promoter (RsrII + MluI) pMB4616 pMB4529 (RsrII + SpeI) Residual backbone & GPD1 From pMB4589 promoter & XPR2 terminator (RsrII + SpeI) pMB4629 pMB4616 (RsrII + MluI) Residual backbone & TEF1 From pMB4590 promoter (RsrII + MluI) pMB4631 pMB4603 (KpnI + NheI) 1.2 kb GPD1 promoter MO4568 & (KpnI + NheI); MO4659 pMB4628 pMB4603 Carp See 1A pMB4637 pMB4629 (NheI + MluI) 1.5 kb hmg1^(trunc )ORF See 1D (XbaI + MluI) pMB4638 pMB4629 carB(i⁻) See 1B pMB4660 pMB4638 (+URA3) carB(i⁻) See 1C pMB4662 pMB4631 (SpeI + XhoI) 1.8 kb URA3 fragment MO4684 & (SpeI + BsaI MO4685 See 1C pMB4683 pMB4662 (Acc65I + MluI) 1.4 kb teflp-GGS1 From pMB4591 fragment (Acc65I + MluI) pMB4692 pMB4662 (Acc65I + MluI) 0.4 kb TEF1 promoter See 1E (Acc65I + NheI); 0.55 kb crtZ ORF (XbaI + MluI) pMB4698 pMB4629 (NheI + MluI) 0.9 kb crtW ORF See 1F (XbaI + MluI) pMB4599 pBluescriptSKII- 1.9 kb carRP gene MO4525 & (EcoRV) MO4541 pMB4606 pBluescriptSKII- 1.9 kb carB gene MO4530 & (EcoRV) MO4542 pMB4613 pMB4599 carRP(i⁻) See text (Acc65I + PpuMI) pMB4619 pBluescriptSKII- carB(i⁻) See text (BamHI + Acc65I)) Certain oligonucleotides referred to in Table 27 above are as follows: MO4471 5′-CTGGGTGACCTGGAAGCCTT (SEQ ID NO: 3) MO4472 5′-AAGATCAATCCGTAGAAGTTCAG (SEQ ID NO: 4) MO4475 5′-AAGCGATTACAATCTTCCTTTGG (SEQ ID NO: 5) MO4476 5′-CCAGTCCATCAACTCAGTCTCA (SEQ ID NO: 6) MO4477 5′-GCATTGCTTATTACGAAGACTAC (SEQ ID NO: 7) MO4478 5′-CCACTGTCCTCCACTACAAACAC (SEQ ID NO: 8) MO4534 5′-CACAAACGCGTTCACTGCGCATCCTCAAAGT (SEQ ID NO: 9) MO4544 5′-CACAATCTAGACACAAATGGATTATAACAGCGCGGAT (SEQ ID NO: 10) MO4566 5′-CACAAACTAGTTTGCCACCTACAAGCCAGAT (SEQ ID NO: 11) MO4568 5′-CACAAGGTACCAATGTGAAAGTGCGCGTGAT (SEQ ID NO: 12) MO4571 5′-CACAAGGTACCAGAGACCGGGTTGGCGG (SEQ ID NO: 13) MO4591 5′-CACAAGCGGCCGCGCTAGCATGGGGATCGATCTCTTATAT (SEQ ID NO: 14) MO4592 5′-CACAAGCGGCCGCGCTAGCGAATGATTCTTATACTCAGAAG (SEQ ID NO: 15) MO4593 5′-CACAAGCGGCCGCACGCGTGCAATTAACAGATAGTTTGCC (SEQ ID NO: 16) MO4659 5′-CACAAGCTAGCTGGGGATGCGATCTCTTATATC (SEQ ID NO: 17) 1A: Production of pMB4628 (teflp-carRP LEU2) encoding phytoene synthase/lycopene cyclase: Intron-containing carRP was amplified from M. circinelloides (ATCC 90680) genomic DNA using MO4525 and MO4541: MO4525 5′-CACAAACGCGTTTAAATGGTATTTAGATTTCTCATT (SEQ ID NO: 18) MO4541 5′-CACAATCTAGACACAAATGCTGCTCACCTACATGGA (SEQ ID NO: 19) and the resulting 1.9 kb fragment was phosphorylated with T4 polynucleotide kinase. The resulting fragment was blunt-end ligated into pBluescriptSKII-cleaved with EcoRV, yielding pMB4599. The 1.9 kb XbaI-MluI fragment from pMB4599 was inserted into NheI- and MluI-cleaved pMB4603, yielding pMB4628. The intron containing nucleic acid coding sequence, and encoded CarRP protein of pMB4628 are as follows:

(SEQ ID NO: 20) atgctgctcacctacatggaagtccacctctactacacgctgcctgtgctgggcgtcctgtectggctgtcgcg gccgtactacacagccaccgatgcgctcaaattcaaatttctgacactggttgccttcacgaccgcctccgcct gggacaactacattgtctaccacaaggcgtggtcctactgccccacctgcgtcaccgctgtcattggctacgtg ccatggaggagtacatgttatcatcatcatgactctgttgaccgtggcattcaccaatctggtgatgcgctggc acctgcacagcttctttatcaggcctgaaacgcccgtcatgcagtccgtcctggtccgtcttgtccccataaca gccttattaatcactgcatacaaggcttgggtaagcaaacaaacaaatgatgtgccgcatcgcattttaatatt aaccattgcatacacagcatttggcggtccctggaaagccactgttctacggatcatgcattttgtggtacgcc tgtccggttttggccttattgtggtttggtgctggcgagtacatgatgcgtcgtccgctggcggtgctcgtctc cattgcgctgcccacgctgtttctctgctgggtcgatgtcgtcgctattggcgccggcacatgggacatttcgc tggccacaagcaccggcaagttcgtcgtgccccacctgcccgtggaggaattcatgttattgcgctaattaata ccgttttggtatttggtacgtgtgcgatcgatcgcacgatggcgatcctccacctgttcaaaaacaagagtcct tatcagcgcccataccagcacagcaagtcgttcctccaccagatcctcgagatgacctgggccttctgtttacc cgaccaagtgctgcattcagacacattccacgacctgtccgtcagctgggacatcctgcgcaaggcctccaagt ccttttacacggcctctgctgtattcccggcgacgtgcgccaagagctcggtgtgctatacgccttttgcagag ccacggacgatctctgcgacaacgagcaggtccctgtgcagacgcgaaaggagcagctgatactgacacatcag ttcgtcagcgatctgtttggccaaaagacaagcgcgccgactgccattgactgggacttttacaacgaccaact gcctgcctcgtgcatctctgccttcaagtcgttcacccgtttgcgccatgtgctggaagctggagccatcaagg aactgctcgacgggtacaagtgggatttggagcgtcgctccatcagggatcaggaggatctcagatattactca gcttgtgtcgccagcagtgttggtgaaatgtgcactcgcatcatactggcccacgccgacaagcccgcctcccg ccagcaaacacagtggatcattcagcgtgcgcgtgaaatgggtctggtactccaatatacaaacattgcaagag acattgtcaccgacagcgaggaactgggcagatgctacctgcctcaggattggcttaccgagaaggaggtggcg ctgattcaaggcggccttgcccgagaaattggcgaggagcgattgctctcactgtcgcatcgcctcatctacca ggcagacgagctcatggtggttgccaacaagggcatcgacaagctgcccagccattgtcaaggcggcgtgcgtg cggcctgcaacgtctatgcttccattggcaccaagctcaagtcttacaagcaccactatcccagcagagcacat gtcggcaattcgaaacgagtggaaattgctatcttagcgtatacaacctttacaccgcgccaattgcgactagt agtaccacacattgcagacagggaaaaatgagaaatctaaataccatttaa; (SEQ ID NO: 21) mlltymevhlyytlpvlgvlswlsrpyytatdalkfkfltlvafttasawdnyivyhkawsycptcvtavigyv pleeymffiimtlltvaftnlvmrwhlhsffirpetpvmqsylvrlypitallitaykawhlavpgkplfygsc ilwyacpvlallwfgageymmaplaylvsialptlflewvdvvaigagtwdislatstgkfvvphlpveefmff alintvlvfgtcaidrtmailhlfknkspyqrpyqhsksflhqilemtwafelpdqvlhsdtfhdlsyswdilr kasksfytasavfpgdvrqelgvlyafcratddlcdneqvpvqtrkeqlilthqfvsdlfgqktsaptaidwdf yndqlpascisafksftrlrhyleagaikelldgykwdlerrsirdqedlryysacvassvgemetriilahad kpasrqqtqwiiqraremglvlqytniardivtdseelgrcylpqdwitekevaliqgglareigeerllslsh rliyqadelmvvankgidklpshcqggvraacnvyasigtklksykhhypsrahvgnskrveiallsvynlyta piatsstthcrqgkmmlnti.

Alternatively, pMB4599 was also used as a template for PCR amplification using MO4318, MO4643, MO4644, and MO4639 and

(SEQ ID NO: 22) MO4318 5′-GTAAAACGACGGCCAGT (SEQ ID NO: 23) MO4643 5′-CACACGGTCTCATGCCAAGCCTTGTATGCAGTGATTAA (SEQ ID NO: 24) MO4639 5′-CCACTGTGTTTGCTGGCGG (SEQ ID NO: 25) MO4644 5′-CACACGGTCTCTGGCATTTGGCGGTCCCTGGAAA producing fragments of 0.5 and 0.95 kb, that were subsequently cleaved with Acc65I and BsaI, and BsaI and PpuMI, respectively. These fragments were ligated to pMB4599 that had been digested with Acc65I and PpuMI, yielding pMB4613, harboring intronless carRP. The 1.85 kb XbaI-Mlul fragment from pMB4613 can be inserted into NheI- and MluI-cleaved pMB4603 to yield pCarRPdelI.

1B: Production of pMB4638 (tef1p-carB ADE1), Encoding Phytoene Dehydrogenase:

Intron-containing carB was amplified from M. circinelloides (ATCC 90680) genomic DNA using MO4530 and MO4542:

(SEQ ID NO: 26) MO4530 5′-CACAAACGCGTTTAAATGACATTAGAGTTATGAAC (SEQ ID NO: 27) MO4542 5′-CACAATCTAGACACAAATGTCCAAGAAACACATTGTC and the resulting 1.9 kb fragment was phosphorylated with T4 polynucleotide kinase and blunt-end ligated into pBS-SKIT-cleaved with EcoRV, yielding pMB4606. pMB4606 was then used as a template for PCR amplification using MO4318 and MO4648, and MO4646 and MO4647, and MO4343 and MO4645:

(SEQ ID NO: 28) MO4318 5′-GTAAAACGACGGCCAGT (SEQ ID NO: 29) MO4648 5′-CACAAGGTCTCAAGCACGCATCCCGGAACTG (SEQ ID NO: 30) MO4646 5′-CACACGGTCTCAGGCATGTCGCCCTACGATGC (SEQ ID NO: 31) MO4647 5′-CACACGGTCTCATGCTTGCACCCACAAAGAATAGG (SEQ ID NO: 32) MO4343 5′-CAGGAAACAGCTATGAC (SEQ ID NO: 33) MO4645 5′-CACACGGTCTCTTGCCCATATACATGGTCTGAAACG producing fragments of 0.4 and 0.85 and 0.7 kb, that were subsequently cleaved with Acc65I and BsaI, and BsaI, and BsaI and BamHI, respectively. These fragments were ligated to pBS-SKII- that had been cut with Acc65I and BamHI, yielding pMB4619, harboring intronless carB. The 1.75 kb XbaI-MluI fragment from pMB4619 was inserted into NheI- and MluI-cleaved pMB4629, yielding pMB4638. The resulting nucleic acid coding sequence and encoded CarB protein of pMB4638 are as follows:

(SEQ ID NO: 34) atgtccaagaaacacattgtcattatcggtgctggcgtgggtggcacggctacagctgctcgtttggcccgcga aggcttcaaggtcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatccatcaccagggcc atcgctttgatcagggcccgtcgctctacctgatgcccaagtactttgaggacgcctttgccgatctggacgag cgcattcaagaccacctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgacggtgagtcgat ccagctgtcgtctgacttgacacgcatgaaggctgaattggaccgcgtggagggcccccttggttttggccgat tcctggatttcatgaaagagacacacatccactacgaaagcggcaccctgattgcgctcaagaagaatttcgaa tccatctgggacctgattcgcatcaagtacgctccagagatctttcgcttgcacctgtttggcaagatctacga ccgcgcttccaagtacttcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatgggcatgt cgccctacgatgcgcctgctgtctacagcctgttgcagtacaccgagttcgctgaaggcatctggtatccccgt ggcggcttcaacatggtggttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatctacaa tgcgcctgttgccaagattaacaccgatgatgccaccaaacaagtgacaggtgtaaccttggaaaatggccaca tcatcgatgccgatgcggttgtgtgtaacgcagatctggtctatgcttatcacaatctgttgcctccctgccga tggacgcaaaacacactggcttccaagaaattgacgtcttatccatttccttctactggtccatgtccaccaag gtgcctcaattggacgtgcacaacatctttttggccgaggcttatcaggagagctttgacgaaatcttcaagga ctttggcctgccttctgaagcctccttctacgtcaatgtgccctctcgcatcgatccttctgctgctcccgacg gcaaggactctgtcattgtcttggtgcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaac tacccggccatggtggacaaggcacgcaagatggtgctggctgtgattgagcgtcgtctgggcatgtcgaattt cgccgacttgattgagcatgagcaagtcaatgatcccgctgtatggcagagcaagttcaatctgtggagaggct caattctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccagcacaaaggattctaccggtcgt tatgataacctattctttgtgggtgcaagcacgcatcccggaactggtgttcccattgtccttgcaggaagcaa gctcacctctgaccaagttgtcaagagattggaaagacgcccaagccaagaaagatcgagatggagaacacgca agcacattggaggagcctgatgctgaatcgacattccctgtgtggttctggttgcgcgctgccttttgggtcat gtttatgttcttttacttcttccctcaatccaatggccaaacgcccgcatcttttatcaataatttgttacctg aagtattccgcgttcataactctaatgtcatttaa; (SEQ ID NO: 35) mskkhiviigagyggtataarlaregfkytyvekndfgggreshhhqghrfdqgpslylmpkyfedafadlder iqdhlellrednnykvhfddgesiqlssditrmkaeldrvegplgfgrfldfmkethihyesgthalkknfesi wdlirikyapeifrlhlfgkiydraskyfkildcmrmaftfqtmymgmspydapavysllqytefaegiwyprg gfnmvvqkleaiakqkydaefiynapvakintddatkqvtgvtlenghiidadavvenadlvyayhnllpperw tqntlaskklisssisfywsmstkvpqldvhniflaeayqesfdeifkdfglpseasfyynypsridpsaapdg kdsvivlypighmksktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavwqskfnlwrgs ilglshdvlqvlwfrpstkdstgrydnlffvgasthpgtgvpivlagskltsdqvvksfgktpkprkiementq apleepdaestfpvwfwfraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi.

1C. Production of pMB4660 (tef1p-carB URA3) Encoding Phytoene Dehydrogenase:

The 4.3 kb XhoI-NotI fragment and the 1.8 kb NotI-SpeI fragment from pMB4638 were ligated to the 1.9 kb BsaI- and SpeI-cleaved URA3 gene generated by PCR amplification of Y. lipolytica genomic DNA using MO4684 and MO4685 to create pMB4660:

(SEQ ID NO: 36) MO4684 5′-CATTCACTAGTGGTGTGTTCTGTGGAGCATTC (SEQ ID NO: 37) MO4685 5′-CACACGGTCTCATCGAGGTGTAGTGGTAGTGCAGTG The resulting nucleic acid coding sequence and encoded CarB(i) protein of pMB4660 are as follows:

(SEQ ID NO: 38) atgtccaagaaacacattgtcattatcggtgctggcgtgggtggcacggctacagctgctcgtttggcccgcga aggcttcaaggtcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatccatcaccagggcc atcgctttgatcagggcccgtcgctctacctgatgcccaagtactttgaggacgcctttgccgatctggacgag cgcattcaagaccacctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgacggtgagtcgat ccagctgtcgtctgacttgacacgcatgaaggctgaattggaccgcgtggagggccccatggttttggccgatt cctggatttcatgaaagagacacacatccactacgaaagcggcaccctgattgcgctcaagaagaatttcgaat ccatctgggacctgattcgcatcaagtacgctccagagatctttcgcttgcacctgtttggcaagatctacgac cgcgcttccaagtacttcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatgggcatgtc gccctacgatgcgcctgctgtctacagcctgttgcagtacaccgagttcgctgaaggcatctggtatccccgtg gcggcttcaacatggtggttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatctacaat gcgcctgttgccaagattaacaccgatgatgccaccaaacaagtgacaggtgtaaccttggaaaatggccacat catcgatgccgatgcggttgtgtgtaacgcagatctggtctatgatatcacaatctgttgcctccctgccgatg gacgcaaaacacactggcttccaagaaattgacgtatatccatttccttctactggtccatgtccaccaaggtg cctcaattggacgtgcacaacatctttttggccgaggcttatcaggagagctttgacgaaatcttcaaggactt tggcctgccttctgaagcctccttctacgtcaatgtgccctctcgcatcgatccttctgctgctcccgacggca aggactctgtcattgtcttggtgcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaactac ccggccatggtggacaaggcacgcaagatggtgctggctgtgattgagcgtcgtctgggcatgtcgaatttcgc cgacttgattgagcatgagcaagtcaatgatcccgctgtatggcagagcaagttcaatctgtggagaggctcaa ttctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccagcacaaaggattctaccggtcgttat gataacctattctttgtgggtgcaagcacgcatcccggaactggtgttcccattgtccttgcaggaagcaagct cacctctgaccaagttgtcaagagattggaaagacgcccaagccaagaaagatcgagatggagaacacgcaagc acctttggaggagcctgatgctgaatcgacattccctgtgtggttctggttgcgcgctgccttttgggtcatgt ttatgttcttttacttcttccctcaatccaatggccaaacgcccgcatcttttatcaataatttgttacctgaa gtattccgcgttcataactctaatgtcatttaa; (SEQ ID NO: 39) mskkhiviigagvggtataarlaregfkvtvvekndfgggreslihhqghrfdqgpslylmpkyfedafadlde riqdhlellrednnykvhfddgesiqlssdlirmkaeldrvegplgfgrfldfmkethihyesgtlialkknfe siwdlirikyapeifrlhlfgkiydraskyfktldcmrmaftfqtmymgmspydapavysllqytefaegiwyp rggfnmvvqkleaiakqkydaefiynapvakintddatkqvtgvtlenghiidadavvenadlvyayhnllppe rwtqntlaskklisssisfywsmstkvpqldvhniflaeayqesfdendfglpseasfyvnvpsridpsaapdg kdsvivlvpighmksktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavwqskfnlwrgs ilglshdvlqvlwfrpstkdstgrydniffvgasthpgtgvpivlagskitsdqvyksfgktpkprkiementq apleepdaestfpvwfwlraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi;

1D. Production of pMB4637 and pTef-HMG Encoding a Truncated HMG1.

For production of a truncated variant of the HMG-CoA reductase gene, which also encodes a 77 amino acid leader sequence derived from S. cerevisiae, the following oligonucleotides are synthesized:

(SEQ ID NO: 40) PRIMER O 5′-TTCTAGACACAAAAATGGCTGCAGACCAATTGGTGA (SEQ ID NO: 41) PRIMER P 5′-CATTAATTCTTCTAAAGGACGTATTTTCTTATC (SEQ ID NO: 42) PRIMER Q 5′-GTTCTCTGGACGACCTAGAGG (SEQ ID NO: 43) MO4658 5′-CACACACGCGTACACCTATGACCGTATGCAAAT. Primers O and P are used to amplify a 0.23 kb fragment encoding Met-Ala followed by residues 530 to 604 of the Hmg1 protein of S. cerevisiae, using genomic DNA as template. Primers Q and MO4658 are used to amplify a 1.4 kb fragment encoding the C-terminal 448 residues of the Hmg1 protein of Y. lipolytica, using genomic DNA as template. These fragments are ligated to the appropriate cloning vector, and the resultant plasmids, designated pOP and pQMO4658, are verified by sequencing. The OP fragment is liberated with XbaI and AseI, and the QMO4658 fragment is liberated with MaeI and MluI. These fragments are then ligated to the ADE1 TEF1p expression vector pMB4629 cut with XbaI and MluI to produce pTefHMG.

Alternatively, the native HMG1 gene from Y. lipolytica may be modified without S. cerevisiae sequences as described in the table above using primers MO4658 (described above) and MO4657, to create pMB4637:

MO4657 (SEQ ID NO: 44) 5′-CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGG. The resulting nucleic acid coding sequence and encoded Hmg^(trunc) protein of pMB4637 are as follows:

(SEQ ID NO: 45) atgacccagtctgtgaaggtggttgagaagcacgttcctatcgtcattgagaagcccagcgagaaggaggag gacacctcttctgaagactccattgagctgactgtcggaaagcagcccaagcccgtgaccgagacccgttctctggacgacctagag gctatcatgaaggcaggtaagaccaagcttctggaggaccacgaggttgtcaagctctctctcgagggcaagcttcctttgtatgctct tgagaagcagcttggtgacaacacccgagctgttggcatccgacgatctatcatctcccagcagtctaataccaagactttagagacc tcaaagcttccttacctgcactacgactacgaccgtgtttttggagcctgttgcgagaacgttattggttacatgcctctccccgttggtgt tgctggccccatgaacattgatggcaagaactaccacattcctatggccaccactgagggttgtcttgttgcctcaaccatgcgaggtt gcaaggccatcaacgccggtggcggtgttaccactgtgcttactcaggacggtatgacacgaggtccttgtgtttccttcccctctctc aagcgggctggagccgctaagatctggcttgattccgaggagggtctcaagtccatgcgaaaggccttcaactccacctctcgatttg ctcgtctccagtctcttcactctacccttgctggtaacctgctgtttattcgattccgaaccaccactggtgatgccatgggcatgaacatg atctccaagggcgtcgaacactctctggccgtcatggtcaaggagtacggcttccctgatatggacattgtgtctgtctcgggtaacta ctgcactgacaagaagcccgcagcgatcaactggatcgaaggccgaggcaagagtgttgttgccgaagccaccatccctgctcac attgtcaagtctgttctcaaaagtgaggttgacgctcttgttgagctcaacatcagcaagaatctgatcggtagtgccatggctggctct gtgggaggtttcaatgcacacgccgcaaacctggtgaccgccatctaccttgccactggccaggatcctgctcagaatgtcgagtctt ccaactgcatcacgctgatgagcaacgtcgacggtaacctgctcatctccgtttccatgccttctatcgaggtcggtaccattggtgga ggtactattttggagccccagggggctatgctggagatgcttggcgtgcgaggtcctcacatcgagacccccggtgccaacgccca acagcttgctcgcatcattgcttctggagttcttgcagcggagctttcgctgtgttctgctcttgctgccggccatcttgtgcaaagtcata tgacccacaaccggtcccaggctcctactccggccaagcagtctcaggccgatctgcagcgtctacaaaacggttcgaatatttgcat acggtcatag; (SEQ ID NO: 46) mtqsvkvvekhypiviekpsekeedtssedsieltvgkqpkpvtetrslddleaimkagktklledhevvk lslegklplyalekqlgdntravgirrsiisqqsntktletsklpylhydydrvfgaccenvigymplpvgvagpmnidgknyhip mattegclvastmrgckainagggvttvltqdgmtrgpcvsfpslkragaakiwldseeglksmrkafnstsrfarlqslhstlag nllfirfrtttgdamgmnmiskgvehslavmvkeygfpdmdivsvsgnyctdkkpaainwiegrgksvvaeatipahivksv lksevdalvelnisknligsamagsvggfnahaanlvtaiylatgqdpaqnvessncitlmsnvdgnllisvsmpsievgtigggt ilepqgamlemlgvrgphietpganaqqlariiasgvlaaelslcsalaaghlvqshmthnrsqaptpakqsqadlqrlqngsnic irs.

1E. Production of pMB4692 (URA3 tef1p-crtZ) Encoding Carotene Hydroxylase.

The following carotene hydroxylase (CrtZ) ORF sequence was synthesized; based on protein sequence of Novosphingobium aromaticivorans, using Y. lipolytica codon bias:

(SEQ ID NO: 47) 5′-ttctagacacaaaa atgggtggagccatgcagaccctcgctgctatcctgatcgtcctcggtacagtgctcgctatggagtttgt cgcttggtcttctcataagtatatcatgcatggcttcggatggggatggcatagagaccatcacgagccccatgagggatttct tgagaagaatgacttatacgccatcgttggcgctgccctctcgatactcatgtttgccctcggctctcccatgatcatgggcgct gacgcctggtggcccggaacctggatcggactcggtgtcctcttctatggtgtcatctataccctcgtgcacgacggtctggtg caccaacgatggtttagatgggtgcctaaacgaggttacgccaaacgactcgtgcaggcccataagctgcaccacgccacc attggcaaggaaggaggcgtctcattcggtttcgtgttcgcccgagatcccgccgttctgaagcaggagcttcgagctcaacg agaagcaggtatcgccgtgctgcgagaggctgtggacggc tagacgcgt. This sequence was cleaved using XbaI and MluI and ligated, along with an Acc65I-NheI TEF1 promoter fragment from pMB4629, to pMB4662 cut with Acc65I and MluI to produce pMB4692. The nucleic acid coding sequence is depicted in bold underline above. The resulting encoded crtZ protein of pMB4692 is as follows:

mggamqtlaailivlgtylamefvawsshkyimhgfgwgwhrdhhephegflekndlyaivgaalsil mfalgspmimgadawwpgtwiglgylfygviytlyhdglyhqrwfrwvpkrgyakrlyqahklhhatigkeggysfgfvfa rdpaylkqelragreagiavlreavdg (SEQ ID NO:82)

1F. Production of pMB4698 (ADE1 tef1p-crtW), Encoding Carotene Ketolase.

The following carotene ketolase (CrtW) ORF sequence was synthesized, based on protein sequence of an environmental sequence isolated from the Sargasso Sea (Genbank accession AACY01034193.1):

(SEQ ID NO: 48) 5′-ttctagacacaaaa atgactcgatctatttcctggccttccacctactggcacctccagccctcctgttcttcttgggtcgcaaacg aattctctcctcaagcccgaaaaggtctcgtcctcgctggtctcattggttccgcttggctgcttactctcggacttggcttttccc ttcccctccatcaaacgagctggcttctcatcggttgtctcgttctccttagatctttcctgcacaccggactttttatcgttgccca tgacgctatgcacgcttctcttgttcctgaccaccctggccttaaccgttggattggacgtgtctgtcttctcatgtatgctggact ctcctacaaaagatgctgccgaaatcaccgtcgacaccaccaagcccctgaaacagttgaagaccctgactaccaacgatg cactaacaacaatatcctcgactggtacgttcactttatgggaaattacctcggatggcaacaattgcttaatctctcttgcgttt ggctcgctctcaccttccgtgtttctgactactctgctcaattcttccacctgctcctifictctgtccttcctctcatcgtctcctcct gtcaactcttcctcgtgggaacctggctgccacaccgacgaggcgctactactcgacccggcgttaccactcgatccctgaac ttccaccctgctctttccttcgctgcttgctaccacttcggttaccaccgtgaacaccatgaatctccctctactccttggttccaa cttcctaaactccgagaaggttctctcatctaa acgcgt. This sequence was cleaved using XbaI and MluI and ligated to pMB4629 cut with NheI and MluI to produce pMB4698. The nucleic acid coding sequence is depicted in bold underline above. The resulting encoded crtW protein of pMB4698 is as follows:

(SEQ ID NO: 83) mtrsiswpstywhlqpscsswvanefspqarkglvlagligsawlltlglgfslplhqtswlligclvllrsflh tglfivahdamhaslvpdhpglnrwigrvcllmyaglsykrccrnhrrhhqapetvedpdyqrctnnnildwyvhfmgnylg wqqllnlscvwlaltfrvsdysaqffhlllfsvlplivsscqlflvgtwlphrrgattrpgvttrslnfhpalsfaacyhfgyhrehhes pstpwfqlpklregsli.

Example 2 Engineering Yarrowia lipolytica for Increased Carotenoid Production

2A. Production of Y. Lipolytica Expressing Geranylgeranylpyrophosphate Synthase and Phytoene Dehydrogenase:

MF350 (MATB ura2-21 leu2-35 ade1) was transformed with pMB4591 (tef1p-GGS1) that had been cleaved upstream of URA5 with SspI; a Ura⁺ transformant carrying the plasmid at the ura2 locus was identified and named MF364. It was subsequently transformed with pMB4638 (tef1p-carB) that had been cleaved at ADE1 with SspI and a prototrophic transformant was chosen that harbored the plasmid at the ade1 locus. This strain was named MF502.

2B. Production of Y. lipolytica Expressing Geranylgeranylpyrophosphate Synthase, Phytoene Dehydrogenase and Phytoene Synthase/Lycopene Cyclase

MF502 was transformed with pMB4628 (tef1p-carRP) that had been treated with SspI. Nine prototrophic colonies were chosen that were uncolored, orange, or very orange on the transformation plate (YNB agar with 1% glucose and 0.1% glutamate [YNBglut]) after two to three days of growth. Two, MF597 and MF600 (the very orange ones), produced greater than 4 mg carotene per g dry cell weight (DCW) after four days of growth in YPD at 30° C. Southern analysis reveals a different single KpnI-HindIII band in genomic DNA from MF597 and MF600, neither of which suggested that homologous integration occurred at leu2-270.

2C. Production of Y. Lipolytica Expressing Phytoene Synthase/Lycopene Cyclase and Phytoene Dehydrogenase:

ATCC201249 (MATA ura3-302 leu2-270 lys8-11) was transformed with SspI-cleaved pMB4628. Hundreds of Leu⁺ colonies were pooled, re-grown, and transformed with pMB4660 (tef1p-carB) that had been cleaved upstream of URA3 with SalI. One colony that was noticeably yellow after 5 days at 30° C. on YNBglut plus 0.6 mM lysine was selected, named MF447, and found to produce 0.2 mg carotene per gram dry cell weight after 4 days of growth in YPD.

MF447 was challenged with 1 g/L 5-fluoroorotic acid and Ura⁻ segregants selected. Surprisingly, they were all found to retain the identical yellow appearance of their parent, implying that the loss of a functional URA3 gene did not coincide with the loss of a functional CarB enzyme. Southern analysis demonstrates that two fragments from a KpnI-HindIII digest of MF447 DNA contain URA3p-hybridizing sequences, only one of which also hybridizes to carB. The other is absent in MF578, the Ura3⁻ segregant chosen for further manipulation. Plasmid rescue and analysis of the DNA sequence encompassing the carRP intron in strains MF447, MF597 (example 2c), and MF600 (example 2c) revealed that exons 1 and 2 were contiguous and were each separated by an intron sequence that lacked the original internal SspI site (present in pMB4628).

2D. Production of Y. lipolytica Expressing Phytoene Synthase/Lycopene Cyclase, Phytoene Dehydrogenase and Geranylgeranylpyrophosphate Synthase:

MF578 was transformed with pMB4683 (tef1p-GGS1) that had been cleaved with SalI (upstream of URA3) or with StuI (within the GGS1 ORF). Ura⁺ Leu⁺ colonies in both cases appeared bright orange on YNBglut+Lys and on YPD, and several produced greater than 4 mg carotene per gram of dry cell weight when grown as above. One, MF633, contained a single copy of the plasmid at the GGS1 locus, as inferred from Southern analysis. The others arose by non-homologous or more complex integrations.

2E. Production of Y. lipolytica Expressing Phytoene Synthase/Lycopene Cyclase, Phytoene Dehydrogenase and Geranylgeranylpyrophosphate Synthase:

MF364 is crossed with MF578, and spores from the resulting diploid are plated on YPD for two to three days at 30° C. Orange Leu⁺ Ade⁻ Ura⁻ colonies are screened for the presence of tefp-carB, tefp-carRP, and tefp-GGS1 by PCR, and for high carotenoid (>4 mg/g dry cell weight) production after growth in YPD liquid medium. Colonies meeting these criteria, as well as displaying resistance to 5-fluorootic acid, an indication that they harbor the ura3-302 allele, are chosen for further studies and hereafter referred to as GBRPua strains. Such a strain is selected for further analysis and modification.

Example 3 Extraction of Carotenoids from Yarrowia lipolytica Cells

Shake-flask testing of generated strains was conducted using YPD medium (1% yeast extract, 2% peptone, 2% glucose). 20 ml cultures in 125 ml flasks were grown at 30° C. Y. lipolytica cells were harvested from 72-96 hour cultures, and extractions were performed to determine carotenoid form and quantity. 1.8 ml of culture was placed into an Eppendorf tube. Cells were pelleted and washed twice with 1 ml H₂O. After the second wash, the resuspended cells were transferred to a pre-weighed snap-cap tube with a hole poked in the top, and the cells were lyophilized overnight. After drying to completion, the tube was weighed in order to calculate dry cell weight. 0.25 ml from the same shake flask culture was placed into a 2 ml screw-cap tube for carotenoid extraction. Cells were pelleted and the supernatant was aspirated. Pelleted cells may be frozen at −80° C. and stored. An equal volume of cubic zirconia beads was added to cell pellets, along with 1 ml ice-cold extraction solvent (a 50/50 v/v mix of hexane and ethyl acetate containing 0.01% butylhydroxytoluene (BHT)). The mixture was then agitated (Mini-BeadBeater-8, BioSpec Products, Inc.) at maximum speed for 5 minutes at 4° C. The mixture was then spun at maximum speed for 1 minute, and the supernatant was collected and deposited in a cold 16 ml glass vial. The remaining cell debris was re-extracted at least three times, without the addition of zirconia beads; all supernatants were pooled in the 16 ml glass vial. Following extraction, the glass vial was spun for 5 minutes at 2000 rpm at 4° C. in a Sorvall tabletop centrifuge, and the supernatant was transferred to a new cold 16 ml glass vial. A Speed Vac was used to concentrate the supernatant (room temperature in dark), and the samples were stored at −20° C. or −80° C. until immediately before HPLC analysis. Prior to HPLC analysis, the samples were resuspended in 1 ml ice-cold solvent and then transferred to a cold amber vial. Throughout the protocol, care was taken to avoid contact with oxygen, light, heat, and acids.

Example 4 Quantification of Carotenoid Production by HPLC

For carotenoid analysis, samples were resuspended in ice-cold extraction solvent (a 50/50 v/v mix of hexane and ethyl acetate containing 0.01% butylhydroxytoluene (BHT)). An Alliance 2795 HPLC (Waters) equipped with a Waters XBridge C18 column (3.5 μm, 2.1×50 mm) and Thermo Basic 8 guard column (2.1×10 mm) was used to resolve carotenoid at 25° C.; authentic carotenoid samples were used as standards. The mobile phases and flow rates are shown below (Solvent A=Ethyl Acetate; Solvent B=Water; Solvent C=Methanol; Solvent D=Acetonitrile). The injection volume was 10 μL. The detector is a Waters 996 photodiode array detector. The retention times for lipophilic molecules include astaxanthin (1.159 min), zeaxanthin (1.335), β-apo-8′-carotenal (2.86 min), ergosterol (3.11 min), lycopene (3.69 min), β-Carotene (4.02 min), and phytoene (4.13 min) Astaxanthin, zeaxanthin, β-apo-8′-carotenal, lycopene and β-Carotene are detected at 475 nm, whereas ergosterol and phytoene were detected at 286 nm.

TABLE 28 Retention Times for Lipophilic Molecules Time Flow (min) (mL/min) % A % B % C % D Curve 0.50 0.0 20.0 0.0 80.0 3.00 1.00 20.0 0.0 0.0 80.0 6 4.50 1.00 80.0 0.0 20.0 0.0 6 5.50 1.00 0.0 0.0 60.0 40.0 6 6.50 1.00 0.0 0.0 80.0 20.0 6 7.50 1.00 0.0 0.0 100.0 0.0 6 8.50 1.00 0.0 0.0 100.0 0.0 6 9.50 1.00 0.0 20.0 0.0 80.0 6 10.50 0.50 0.0 20.0 0.0 80.0 6

Example 5 Expression of a Truncated Form of HMG-coA Reductase Results in Increased Carotenoid Production

In order to increase carotenoid production, carbon flow through the isoprenoid pathway is enhanced by introducing a truncated variant of the HMG-CoA reductase gene.

In one approach, a truncated variant of the HMG-CoA reductase gene which also encodes a 77 amino acid leader sequence derived from S. cerevisiae Hmg1 is introduced into a GRPBua strain (described in Example 2E above). Plasmid pTefHMG can be cleaved with SnaBI, BbvCI, or Bsu36I to direct integration at the ade1 locus, or with BamHI to direct integration at the HMG1 locus, or with EcoRV to promote random integration, in the GRPBua strains, restoring them to adenine prototrophy. Resulting Ade⁺ transformants are screened for increased carotenoid production.

Alternatively, the native HMG1 gene from Y. lipolytica may be modified without S. cerevisiae sequences as described in Example 1D above, to create pMB4637. This plasmid can be digested as described for pTefHMG and transformed into GRPBua strains, and resulting transformants screened as described for increased carotenoid production.

In still another approach, a truncated variant of the N. crassa HMG-CoA reductase gene may be utilized and introduced into Y. lipolytica strains. In order to generate a plasmid suitable for expression of the heterologous HMG-CoA reductase, p641P (Yeast 2001; 18 (2001): 97-113) is modified by replacing the ICL1 promoter with the GPD promoter, and by the addition of sequences conferring resistance to phleomycin. Y. lipolytica genomic DNA is amplified with two primers.

GPDdist: (SEQ ID NO: 49) 5′ CACACGGTacctgtaggttgggttgggtg GPDprox: (SEQ ID NO: 50) 5′ CACACGGATCCtgtttaattcaagaatgaatatagagaagagaag, and the resulting fragment (0.7 kb) is cleaved with BamHI and KpnI, and ligated to BamHI- and KpnI-cleaved p641P, creating the plasmid “p641 Pgpd”. The ble gene under the control of the A. nidulans GPD promoter is then excised from pBCphleo (Silar, Fungal Genetics Newsletter 42:73) as a 3.2 kb BclI-BamHI fragment and inserted into the unique BamHI site of “p641 Pgpd”, in the orientation that preserves the BamHI site proximal to the GPD promoter, to create “p641 Pgpdble”,

N. crassa genomic DNA is amplified with two primers:

Neuhmg fwd: (SEQ ID NO: 51) 5′ CACACGGATCCACATCAACAatggcatctgccacccttcccc Neuhmg rev: (SEQ ID NO: 52) 5′ CACACGGATCcaagtgctgacgcggaacttg, and the resulting fragment is cleaved with BamHI and inserted into BamHI-digested “p641 Pgpdble” in the correct orientation. The resulting plasmid, “pZg”, contains sequences encoding a truncated cytosolic catalytic domain of hydroxymethylglutaryl-CoA reductase from N. crassa (Genbank accession: XP_(—)324892) under the control of the constitutive GPD promoter. This plasmid can be introduced into the Y. lipolytica strain created in Example 2E above, and transformants are selected by their resistance to phleomycin (100 μg/ml). Resulting transformants are tested for β-carotene production, as described above.

Example 6 Introduction of Heterologous Carotene Hydroxylase and Carotene Ketolase Genes into Y. lipolytica Strains Producing Carotenoid for Production of Astaxanthin

For introduction of carotene hydroxylase and carotene ketolase into carotenoid producting Y. lipolytica, pMB4692 and pMB4698, described as in Example 1E and 1F above, can be sequentially introduced into the GRPBua strain (described in Example 2E). For the introduction of pMB4692, the plasmid may be cleaved with SalI or BsrGI to direct integration at the ura3 locus, or with XbaI to promote random integration, selecting for uracil prototrophy. GRPBua Ura⁺ transformants harboring pMB4692 are screened for zeaxanthin production in YPD. Zeaxanthin-producing cells are transformed with pMB4698 (which can be cleaved with PpuMI, SspI or BbvCI to direct integration at the ade1 locus, or with EcoRV to promote random integration) and prototrophic colonies are screened for astaxanthin production.

Alternatively, the order of plasmid transformation may be reversed wherein pMB4698 is transformed first and transformants are selected for adenine prototrophy. GRPBua Ade⁺ transformants harboring pMB4698 are screened for canthaxanthin production. Canthaxanthin-producing GRPBua[pMB4698] cells are transformed with pMB4692 and prototrophic colonies are screened for astaxanthin production.

In another approach, the carotenoid ketolase and carotenoid hydroxylase genes from P. marcusii can be introduced into the strains described in Example 2 above, in order to convert β-carotene into astaxanthin. P. marcusii genomic DNA is amplified with two primers.

CrtZfwd: (SEQ ID NO: 53) 5′ CACACCGTCTCAAatgaccaatttcctgatcgtcgtc CrtZrev: (SEQ ID NO: 54) 5′ CACACAGATCtcacgtgcgctcctgcgcc, and the resulting fragment is cleaved with BsmBI, modified with the Klenow fragment of DNA polymerase, and cleaved with BglII. This fragment is inserted into PmlI- and BamHI-cleaved pINA1269 (J. Mol. Microbiol. Biotechnol. 2 (2000): 207-216), containing the hp4d promoter, the XPR2 terminator, the selectable LEU2 gene, and sequences necessary for selection and propagation in E. coli. The resulting plasmid “pA” contains sequences encoding carotene hydroxylase from P. marcusii (crtZ gene) (Genbank accession: CAB56060.1) under the control of the hp4d promoter.

“pYEG1TEF” is modified by substituting the LIP2 terminator for the XPR2 terminator as follows. pINA1291 is digested with AvrII, modified with the Klenow fragment of DNA polymerase, and cleaved with EcoRI, and the small LIP2t containing fragment is ligated to “pYEG1TEF” that has been digested with SacII, modified with T4 DNA polymerase in the presence of dNTP, and cleaved with EcoRI. The resulting plasmid is named “pYEG1TEF-LIP2t”.

In order to amplify the carotenoid ketolase gene, P. marcusii genomic DNA is amplified with two primers.

CrtWfwd: (SEQ ID NO: 55) 5′ CACACCCTAGGCCatgagcgcacatgccctgc CrtWrev: (SEQ ID NO: 56) 5′ CACACAAGCTTtcatgcggtgtcccccttg, and the resulting fragment is cleaved with AvrII and HindIII, and inserted into AvrII- and HindIII-cleaved “pYEG1TEF-LIP2t”. The resulting plasmid, “pBt”, contains sequences encoding the carotene ketolase (crtW gene) (Genbank accession: CAB56059.1) under the control of the constitutive TEF1 promoter.

In order to combine the two expression cassettes into a single plasmid, “pBt” is cleaved with ClaI, modified with the Klenow fragment of DNA polymerase, and cleaved with EcoRI, and the crtW-containing fragment is isolated, mixed with the phosphorylated oligonucleotide adaptor pair:

(SEQ ID NO: 57) 5′ AATTCGCGGCCGCT and (SEQ ID NO: 58) 5′ AGCGGCCGCG, cleaved with Nod, and ligated to NotI-digested “pA”. The resulting plasmid, “pABt”, contains both the TEF1p/crtW/LIP2t cassette and the hp4d/crtZ/XPR2t cassette as well as the selectable LEU2 gene. “pABt” can be introduced into the Y. lipolytica strain described above in Example 4 (TEF1p/a1-1/XPR2t; hp4d/carRP/LIP2t; GPDp/HMGR_(trunc)), and transformants selected for leucine prototrophy.

Example 7 Partial Inactivation of Y. lipolytica ERG9 Gene Encoding Squalene Synthase Results in Increased Carotenoid Production

7A. In order to partially inactivate the ERG9 gene encoding squalene synthase, the neighboring FOL3 gene is disrupted, resulting in a folinic acid requirement. This strain is then transformed with a mutagenized fragment of DNA partially spanning the two genes, and For transformants are screened for decreased squalene synthase activity.

The following oligonucleotides are synthesized:

PRIMER K (SEQ ID NO: 59) 5′-CCTTCTAGTCGTACGTAGTCAGC; PRIMER L (SEQ ID NO: 60) 5′-CCACTGATCTAGAATCTCTTTCTGG and used to amplify a 2.3 kb fragment from Y. lipolytica genomic DNA spanning most of the FOL3 gene, using Pfu polymerase. The resulting fragment is cleaved with XbaI and phosphorylated, then ligated into pBluescriptSK⁻ that has been cleaved with KpnI, treated with T4 DNA polymerase (T4pol) in the presence of dNTPs, and subsequently cleaved with XbaI. The resultant plasmid, designated pBS-fol3, is then cleaved with Acc65I and EcoRI, treated with T4pol as above, and ligated to the 3.4 kb EcoRV-SpeI ADE1 fragment (treated with T4pol) from pMB4529.

The resulting plasmid, pBSfol3Δade, can be cleaved with BsiWI and XbaI to liberate a 5.5 kb fragment that is used to transform the GRBPua strains described above to adenine prototrophy. Resulting Ade⁺ transformants are screened for a folinic acid requirement, and for homologous integration by PCR analysis.

Strains that harbor the resultant fol3ΔADE1 allele can be transformed with a 3.5 kb DNA fragment generated by mutagenic PCR amplification using the primers:

PRIMER M (SEQ ID NO: 61) 5′-GGCTCATTGCGCATGCTAACATCG; PRIMER N (SEQ ID NO: 62) 5′-CGACGATGCTATGAGCTTCTAGACG, and Y. lipolytica genomic DNA as template. The resulting fragment containing the N-terminal three-quarters of the FOL3 ORF and the C-terminal nine-tenths of the ERG9 ORF is used to transform strains. The resulting Fol⁺ Ade⁻ transformants are screened for decreased squalene synthase activity by sensitivity to agents such as zaragozic acid, itraconazole, or fluconazole. Additionally, the resulting transformants are screened for increased carotenoid production.

7B. Alternatively, the PCR fragment produced in 7A could be cloned and altered in such a way as to remove the 3′-untranslated region of ERG9 gene. Replacement of the fol3ΔADE1 disruption by this fragment results in decreased expression of squalene synthase [Schuldiner et al. (2005), Cell 123:507-519][Muhlrad and Parker (1999), RNA 5:1299-1307], which can be confirmed as in 7A. This approach may also be used in a Fol⁺ Ade⁻ strain, using the ADE1 marker to disrupt the ERG9 3′-UTR.

7C. In still another approach, partially defective ERG9 alleles can be identified in S. cerevisiae using plasmid shuffling techniques [Boeke et al. (1987), Methods Enzymol. 154:164-175], and using drug sensitivities as a phenotype. Defective genes can be transferred to Y. lipolytica using standard molecular genetic techniques.

Example 8 Treatment of Y. lipolytica Strains Producing Carotenoid with Inhibitor of an Isoprenoid Biosynthesis Competitor Polypeptide Results in Increased Carotenoid Production

Cultures produced in Example 2 are treated with the squalene synthase inhibitor, zaragozic acid (zaragozic acid at 0.5 μM) and monitored for β-carotene production, as described above.

Example 9 Constructing an Oleaginous Strain of Saccharomyces Cerevisiae

The genes encoding the two subunits of ATP-citrate lyase from N. crassa, the AMP deaminase from Saccharomyces cerevisiae, and the cytosolic malic enzyme from M. circinelloides are overexpressed in S. cereviseae strains in order to increase the total lipid content. Similar approaches to enhance lipid production could be employed in other host organisms such as Xanthophyllomyces dendrorhous (Phaffia rhodozyma), using the same, homologous, or functionally similar oleaginic polypeptides.

Qiagen RNAEasy kits (Qiagen, Valencia, Calif.) are used to prepare messenger RNA from lyophilized biomass prepared from cultures of N. crassa. Subsequently, RT-PCR is performed in two reactions containing the mRNA template and either of the following primer pairs.

acl1: (SEQ ID NO: 63) 1fwd: 5′ CACACGGATCCTATAatgccttccgcaacgaccg (SEQ ID NO: 64) 1rev: 5′ CACACACTAGttaaatttggacctcaacacgaccc acl2: (SEQ ID NO: 65) 2fwd: 5′ CACACGGATCCAATATAAatgtctgcgaagagcatcctcg (SEQ ID NO: 66) 2rev: 5′ CACACGCATGCttaagcttggaactccaccgcac

The resulting fragment from the acl1 reaction is cleaved with SpeI and BamHI, and that from the acl2 reaction is cleaved with BamHI and SphI, and both are ligated together into YEp24 that has been digested with NheI and SphI, creating the plasmid “p12”. The bi-directional GAL1-10 promoter is amplified from S. cerevisiae genomic DNA using the primers.

gal10: (SEQ ID NO: 67) 5′ CACACGGATCCaattttcaaaaattcttactttttttttggatggac gal1: (SEQ ID NO: 68) 5′ CACACGGATCCttttttctccttgacgttaaagtatagagg, and the resulting 0.67 kb fragment is cleaved with BamHI and ligated in either orientation to BamHI-digested “p12” to create “p1gal2” and “p2gal1”, containing GAL1-acl1/GAL10-acl2 and GAL10-acl1/GAL1-acl2, respectively (Genbank accession: acl1: CAB91740.2; acl2: CAB91741.2).

In order to amplify the S. cereviseae gene encoding AMP deaminase and a promoter suitable for expressing this gene, S. cerevisiae genomic DNA is amplified using two primer pairs in separate reactions:

AMD1 ORF: AMD1FWD: (SEQ ID NO: 69) 5′ CACACGAGCTCAAAAatggacaatcaggctacacagag AMD1rev: (SEQ ID NO: 70) 5′ CACACCCTAGGtcacttttcttcaatggttctcttgaaattg GAL7p: gal7prox: (SEQ ID NO: 71) 5′ CACACGAGCTCggaatattcaactgtttttttttatcatgttgatg gal7dist: (SEQ ID NO: 72) 5′ CACACGGAtccttcttgaaaatatgcactctatatcttttag, and the resulting fragment from the AMD1 reaction (2.4 kb) is cleaved with Sad and AvrII, and that from the GAL7 reaction (0.7 kb) is cleaved with BamHI and SphI, and both are ligated together into YEp13 that has been digested with NheI and BamHI, creating the plasmid “pAMPD”. This plasmid carries the S. cerevisiae gene, AMD1, encoding AMP deaminase, under the control of the galactose-inducible GAL7 promoter.

Messenger RNA is prepared from lyophilized biomass of M. circinelloides, as described above, and the mRNA template is used in a RT-PCR reaction with two primers:

MAEfwd: (SEQ ID NO: 73) 5′ CACACGCTAGCTACAAAatgttgtcactcaaacgcatagcaac MAErev: (SEQ ID NO: 74) 5′ CACACGTCGACttaatgatctcggtatacgagaggaac, and the resulting fragment is cleaved with NheI and SalI, and ligated to XbaI- and XhoI-digested pRS413TEF (Mumberg, D. et al. (1995) Gene, 156:119-122), creating the plasmid “pTEFMAE”, which contains sequences encoding the cytosolic NADP⁺-dependant malic enzyme from M. circinelloides (E.C. 1.1.1.40; mce gene; Genbank accession: AY209191) under the control of the constitutive TEF1 promoter.

The plasmids “p1gal2”, “pAMPD”, and “pTEFMAE” are sequentially transformed into a strain of S. cereviseae to restore prototrophy for uracil (“p1gal2”), leucine (“pAMPD”), and histidine (“pTEFMAE”) (Guthrie and Fink Methods in Enzymology 194:1-933, 1991). The resulting transformants are tested for total lipid content following shake flask testing in either synthetic complete (SC) medium lacking uracil, leucine and histidine, as described in Example 3, or in a 2-step fermentation process. In the 2-step process, 1.5 ml of cells from an overnight 2 ml roll tube culture containing SC medium lacking uracil, leucine and histidine are centrifuged, washed in distilled water, and resuspended in 20 ml of a nitrogen-limiting medium suitable for lipid accumulation (30 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L NH₄Cl, 7 g/L KH₂PO₄, 5 g/L Na₂HPO₄-12H₂O, 1.5 g/L MgSO₄-7H₂O, 0.08 g/L FeCl₃-6H₂O, 0.01 g/L ZnSO₄-7H₂O, 0.1 g/L CaCl₂-2H₂O, 0.1 mg/L MnSO₄-5H₂O, 0.1 mg/L CuSO₄-5H₂O, 0.1 mg/L Co(NO₃)₂-6H₂O; pH 5.5 (J Am Oil Chem Soc 70:891-894 (1993)).

Intracellular lipid content of the modified and control S. cerevisiae strains is analyzed using the fluorescent probe, Nile Red (J Microbiol Meth (2004) 56:331-338). In brief, cells diluted in buffer are stained with Nile Red, excited at 488 nm, and the fluorescent emission spectra in the wavelength region of 400-700 nm are acquired and compared to the corresponding spectra from cells not stained with Nile Red. To confirm results from the rapid estimation method, the total lipid content is determined by gas chromatographic analysis of the total fatty acids directly transmethylesterified from dried cells, as described (Appl Microbiol Biotechnol. 2002 November; 60(3):275-80). Non-transformed S. cerevisiae strains produce 6% and 10% total lipid (dry cell weight basis) after growth in YPD and lipid accumulation medium, respectively. Yeast strains expressing the multiple oleaginic polypeptides produce 17% and 25% total lipid following growth in YPD and lipid accumulation medium, respectively.

Example 10 Introduction of Heterologous Carotene Hydroxylase into Y. lipolytica Strains Producing Carotenoid for Production of Zeaxanthin

MF578 (tef-carRP tef-carB) was transformed with pMB4692 that had been cleaved with SalI. Several Ura⁺ colonies inferred to contain tef-crtZ by PCR analysis were able to produce zeaxanthin in YPD shake flasks, and in one case, all of the carotene was depleted.

The following tables are referenced throughout the description:

TABLE 1 Examples of acetyl-CoA carboxylase polypeptides. Genbank Row ACCESSION Genbank GI 1 XP_410263 49097606 2 XP_329580 32418204 3 XP_386756 46124405 4 XP_367702 39972623 5 XP_501721 50548503 6 EAK99708 46440402 7 XP_457211 50413128 8 NP_982612 45184894 9 XP_449236 50293649 10 NP_593271 19114183 11 NP_014413 6324343 12 XP_455355 50310667 13 T42531 11272737 14 AAA20073 171504 15 EAL20176 50257469 16 XP_571316 58268320 17 XP_402244 49076566 18 S60200 2133343 19 BAA24410 2804173 20 P32874 1708192 21 S55089 7438088 22 NP_990836 45382859 23 CAE01471 32526576 24 AAR37018 40019048 25 NP_001 . . . 57164283 26 NP_776649 27806341 27 CAI25271 56205878 28 XP_109883 51828611 29 NP_942134 38679971 30 NP_942131 38679960 31 NP_942135 38679974 32 NP_942136 38679977 33 AAP94122 33112885 34 NP_071529 11559962 35 2006242A 740964 36 AAS13685 42405896 37 NP_598665 48976025 38 Q13085 2493311 39 XP_548250 57091783 40 XP_314071 58385597 41 CAG08536 47226520 42 NP_724636 24586460 43 NP_610342 24586458 44 NP_001084 4501855 45 NP_446374 16758804 46 EAL63219 60465120 47 NP_921034 37533464 48 T07084 7438099 49 AAP78896 32264940 50 AAO62903 29123370 51 BAA07012 1100253 52 AAL02056 15558947 53 AAG40563 11869927 54 D86483 25293894 55 T07920 7438090 56 A57710 2130099 57 AAO62902 29123376 58 2208491A 1588584 59 T09538 7438102 60 CAC19875 12057067 61 AAP78897 32264942 62 T02235 7438095 63 AAG40564 11869928 64 E86483 25293893 65 CAC84161 20975574 66 T07081 7438097 67 CAC19876 12057069

TABLE 2 Examples of pyruvate decarboxylase polypeptides. Genbank Row ACCESSION Genbank GI 1 1QPBB 7245977 2 CAA54522 871533 3 1PYDB 515237 4 CAA28380 4109 5 1PVDB 1127233 6 CAA33709 4114 7 AAN77243 25992752 8 NP_013235 6323163 9 Q6FJA3 57012668 10 S36363 486942 11 Q12629 52788279 12 AAP75898 37359468 13 S70684 2131152 14 NP_011601 6321524 15 AAQ73618 34500072 16 NP_983270 45185554 17 AAF78895 8745337 18 CAB65554 6689662 19 AAP75899 37359470 20 NP_982469 45184751 21 CAA97091 1945321 22 S50700 1086157 23 XP_446491 50288125 24 XP_462338 50427451 25 AAC03164 17066784 26 EAK96569 46437219 27 XP_457131 50412425 28 AAC03165 2734883 29 XP_459224 50421349 30 CAH56494 52673248 31 XP_502647 50550349 32 NP_010203 6320123 33 BAA04886 1786148 34 XP_449074 50293325 35 EAL04098 46444826 36 CAD60727 27803024 37 T38759 25777585 38 XP_331173 32421459 39 NP_594083 19114995 40 XP_401609 49075036 41 XP_390010 46136637 42 XP_409025 49095128 43 NP_984350 45188127 44 AAD16178 4323053 45 P87208 2501326 46 EAL18331 50255598 47 XP_567475 58260130 48 AAM73540 21666011 49 AAM73539 21666009 50 XP_502508 50550071 51 CAA93158 1177659 52 XP_412533 49123327 53 P51844 1706333 54 XP_455842 50311631 55 CAA61155 3688422 56 XP_444902 50284947 57 CAA47319 4118

TABLE 3 Examples of isocitrate dehydrogenase polypeptides. Genbank Row ACCESSION Genbank GI 1 O13285 3023996 2 EAK91676 46432179 3 O13285 3023996 4 EAK94305 46434909 5 XP_451683 50303483 6 XP_459772 50422415 7 O13294 27805482 8 XP_460289 50423413 9 XP_390523 46137663 10 XP_367343 39971905 11 XP_323176 32405126 12 XP_445447 50286037 13 AAK76730 15027826 14 NP_010217 6320137 15 NP_984921 45190667 16 AAK76731 15027827 17 P79089 3023999 18 NP_013275 6323203 19 XP_407136 49091350 20 NP_982520 45184802 21 XP_446953 50289047 22 XP_445184 50285511 23 XP_455638 50311227 24 AAA64516 736722 25 NP_970434 42525054 26 AAT93173 51013759 27 XP_569233 58264154 28 XP_569234 58264156 29 XP_403726 49080406 30 XP_503571 50552322 31 XP_458151 50428131 32 O13302 13124301 33 XP_409927 49096934 34 XP_385909 46122711 35 XP_365293 39967489 36 NP_983873 45187650 37 XP_455266 50310493 38 NP_594397 19115309 39 XP_324955 32408949 40 CAE81942 38636405 41 NP_014361 6324291 42 XP_446479 50288101 43 XP_567378 58259936 44 XP_398944 49069310 45 XP_502479 50550013 46 EAK96238 46436883 47 EAK96305 46436951 48 XP_461797 50426401 49 XP_328403 32415850 50 CAF31997 42820684 51 XP_389756 46136129 52 XP_363786 39952139 53 AAL73035 18463935 54 XP_405140 49086142 55 NP_595203 19111995 56 NP_014779 6324709 57 XP_447564 50290265 58 NP_985684 45198655 59 XP_566837 58258849 60 XP_454086 50308171 61 XP_398943 49069308

TABLE 4 Examples of ATP-citrate lyase polypeptides. Genbank Row ACCESSION Genbank GI 1 XP_327071 32413182 2 O93988 30912679 3 XP_370222 39977669 4 XP_406573 49090008 5 XP_504787 50554757 6 Q9P7W3 30912748 7 XP_398620 49068662 8 NP_596202 19112994 9 XP_567460 58260100 10 NP_001008 56118260 11 XP_418154 50760837 12 AAH84253 54038148 13 NP_942127 38569423 14 NP_001087 38569421 15 P53396 20141248 16 AAL34316 17028103 17 NP_001002 50540366 18 AAH84776 54311201 19 S21173 105392 20 AAT94429 51092031 21 AAD34754 28372804 22 AAH21502 18204829 23 XP_319323 58392375 24 NP_725514 24653990 25 EAL26601 54637198 26 CAE56725 39579419 27 CAE64663 39593194 28 XP_511495 55645405 29 CAF95829 47210997 30 AAO22565 27754223 31 AAL33788 17065616 32 CAB46077 5304837 33 CAF96044 47204726 34 AAK13318 13160653 35 AAQ75159 34558815 36 AAQ75128 34558783 37 XP_537640 57091075 38 XP_327069 32413178 39 CAB76164 7160184 40 XP_370223 39977671 41 XP_386215 46123323 42 CAA10666 7159697 43 XP_406572 49090004 44 XP_503231 50551515 45 NP_593246 19114158 46 XP_398620 49068662 47 XP_567460 58260100 48 AAT94429 51092031 49 NP_725514 24653990 50 AAD34754 28372804 51 EAL26601 54637198 52 XP_319323 58392375 53 AAH84776 54311201 54 BAB00624 9229902 55 NP_001008 56118260 56 AAH84253 54038148 57 AAH56378 38614162 58 NP_001087 38569421 59 NP_942127 38569423 60 P53396 20141248 61 XP_511495 55645405 62 NP_058683 8392839 63 NP_001002 50540366 64 S21173 105392 65 NP_508280 17551266 66 CAE64663 39593194 67 CAE56725 39579419 68 NP_506267 17557344 69 XP_537640 57091075 70 CAF96059 47204551 71 F96633 25404292 72 AAM91141 22136126 73 NP_849634 30681854 74 AAO23582 27764922 75 AAM65078 21593129 76 CAC86996 15919089 77 AAQ75158 34558814 78 AAQ75127 34558782

TABLE 5 Examples of malic enzyme polypeptides. Genbank Row ACCESSION Genbank GI 1 NP_012896 6322823 2 XP_448858 50292851 3 XP_454793 50309563 4 NP_986598 45201028 5 XP_460887 50424595 6 EAK97738 46438407 7 XP_504112 50553402 8 XP_330094 32419237 9 XP_380981 46107844 10 XP_411070 49102552 11 XP_362875 39946676 12 NP_587760 19075260 13 NP_978189 42780942 14 YP_035982 49481098 15 YP_027934 49184682 16 YP_018438 47527089 17 ZP_002365 47565532 18 YP_083209 52143619 19 XP_571672 58269032 20 NP_391586 16080758 21 YP_092693 52786864 22 NP_831516 30019885 23 YP_093460 52787631 24 YP_081030 52082239 25 NP_822689 29828055 26 O34389 33517449 27 EAL19111 50256386 28 NP_825047 29830413 29 ZP_002340 47096498 30 NP_928837 37525493 31 NP_230833 15641201 32 NP_934257 37679648 33 NP_761613 27366085 34 AC1314 25283688 35 YP_055602 50842375 36 YP_095310 52841511 37 ZP_002315 47093832 38 AC1686 25283689 39 YP_126594 54294179 40 YP_123567 54297198 41 EAJ76260 44510091 42 YP_114273 53803890 43 NP_797637 28898032 44 YP_040250 49483026 45 ZP_001276 53693400 46 YP_044961 50083451 47 YP_128226 54295811 48 NP_719387 24375344 49 XP_572853 58271394 50 NP_252161 15598667 51 ZP_001368 46164263 52 YP_125345 54298976 53 NP_793695 28871076 54 YP_096964 52843165 55 EAH92280 44245125 56 YP_154988 56459707 57 EAI68195 44354928 58 YP_070054 51595863 59 YP_133025 54303032 60 NP_969623 42524243 61 NP_856009 31793516 62 DECARBOXY ATING)) 63 NP_935035 37680426 64 YP_050922 50121755 65 E70705 7431223 66 NP_216848 57116971 67 DECARBOXY ATING)) 68 YP_143786 55980489 69 YP_130202 54309182 70 NP_415996 16129438 71 NP_819843 29654151 72 NP_753809 26247769 73 NP_707611 56479957 74 F85728 25283682 75 YP_163690 56552851 76 YP_150562 56413487 77 NP_720610 24378655 78 NP_460525 16764910 79 ZP_003193 48865537 80 NP_784797 28377905 81 T13496 7431227 82 AAV65766 55793550 83 A97096 25283683 84 YP_193951 58337366 85 H97096 25283684 86 ZP_003237 48870993 87 ZP_001460 41689468 88 D86737 25283676 89 ZP_002870 48825851 90 ZP_001439 34762975 91 1922245A 737262 92 YP_169914 56708018 93 YP_055027 50841800 94 ZP_000625 23023297 95 NP_296302 15807565 96 NP_285599 15807938 97 YP_132069 54302076 98 CAA50716 467569 99 ZP_002906 48833596 100 ZP_003155 48861632 101 NP_773109 27381580 102 AAQ95658 37622953 103 CAC19505 56204311 104 AAH80660 51873855 105 P40927 729986 106 AAT02533 46850200 107 BAC37086 26346875 108 T02763 7431235 109 XP_387367 46125627 110 AAC50613 1465733 111 CAA39421 669118 112 CAA39420 669117 113 NP_032641 6678912 114 CAA39419 581228 115 AAB01380 1335389 116 JC4160 1085347 117 E96828 25283677 118 BAD87910 57899974 119 EAJ77083 44511304 120 P13697 266504 121 NP_036732 7106353 122 YP_065939 51246055 123 CAC18164 16944467 124 XP_322953 32404680 125 AAK91502 18460985 126 AAQ88396 37147841 127 NP_001003 57525624 128 1GQ2P 21465488 129 AAO26053 28195290 130 AAH84250 54038006 131 XP_362590 39946106 132 AAH03287 13096987 133 Q29558 2497785 134 XP_532217 57094622 135 P28227 126734 136 NP_496968 17537199 137 NP_914533 34906372 138 AAD10504 4096786 139 AAO67523 50897495 140 P43279 1170871 141 AAK83074 15077109 142 AAP33011 30575690 143 AAN86690 27357017 144 P78715 41017288 145 AAP32204 30526303 146 AAV31249 54287505 147 T06402 7431232 148 Q99KE1 55583978 149 XP_399922 49071266 150 P36444 547886 151 AAO30034 28059162 152 AAK83073 15077107 153 NP_002387 4505145 154 AAA33487 168528 155 BAA74735 4239891 156 NP_989634 45383538 157 1GZ3D 31615316 158 AAW56450 57791240 159 AAT02534 46850202 160 S29742 422339 161 1O0SB 34811253 162 P27443 126732 163 T06401 7431231 164 AAL16175 16226466 165 AAF73006 8118507 166 AAK97530 15420975 167 EAI90348 44385841 168 P51615 1708924 169 AAA19575 169327 170 S43718 1084300 171 P34105 1346485 172 AAS38597 42733630 173 BAC54101 27530932 174 AAT02535 46850204 175 CAB66003 6706333 176 AAH84860 54311418 177 CAA39422 669119 178 NP_916713 34910732 179 CAA56354 510876 180 DEFBC 7427668 181 JC5967 7431234 182 NP_197960 15239517 183 NP_651959 21356279 184 CAB64263 6634090 185 BAB20887 54606800 186 EAL27424 54638022 187 NP_006671 5729920 188 AAB08874 1561774 189 1PJLH 33358128 190 1GZ4D 22218682 191 1QR6B 5822327 192 1PJ3D 39654475 193 P22178 126736 194 XP_410305 49097690 195 AAH22472 18490280

TABLE 6 Examples of AMP deaminase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAA34420 171053 2 XP_446684 50288509 3 NP_983153 45185436 4 XP_453337 50306727 5 EAL02322 46443037 6 XP_460211 50423261 7 XP_503822 50552824 8 XP_413009 49131023 9 XP_360256 39941438 10 XP_381547 46108978 11 XP_330167 32419447 12 CAB97316 16945394 13 T50996 11359582 14 NP_595153 19111945 15 EAL22226 50259553 16 XP_402237 49076548 17 CAA62797 995562 18 AAF65407 7638159 19 XP_537039 57088163 20 AAH49119 29145073 21 XP_569691 58265070 22 AAD56303 5922018 23 NP_004028 21264318 24 A44313 345738 25 CAI19307 56206061 26 AAA62126 644509 27 CAI19305 56206059 28 XP_310497 58424203 29 CAI19306 56206060 30 AAC50308 608499 31 CAG06825 47229629 32 NP_727741 45551453 33 NP_727739 45551452 34 NP_727740 24641890 35 AAN09337 22832227 36 T01259 7484807 37 XP_506591 51963676 38 NP_850294 30687456 39 CAG07509 47228777 40 NP_494974 32564190 41 T15771 7497030 42 CAE59064 39596837 43 NP_494973 32564194 44 BAA06505 1321635 45 NP_000471 4502079 46 S68147 2134756 47 AAH56380 38614134 48 O08739 2494043 49 NP_113732 13928736 50 O09178 2494044 51 XP_420973 50747746 52 NP_956142 41054127 53 CAG01709 47222742 54 NP_957187 41053780 55 XP_392957 48104570 56 AAH07183 13938134 57 CAG05605 47220579 58 NP_620231 20302047 59 XP_540247 57098851 60 CAF99638 47230445 61 XP_513671 55587796 62 CAI18828 56203368 63 CAI18829 56203369 64 CAI18830 56203370 65 EAA19931 23484684 66 CAH99706 56500932 67 XP_131103 38076931 68 CAH77387 56523366

TABLE 7 Examples of acetoacetyl-CoA thiolase polypeptides. Genbank Row ACCESSION Genbank GI 1 P10551 135758 2 Q04677 418002 3 Q12598 34925109 4 T10247 7433657 5 T42741 11257345 6 AAL18924 16417944 7 AAM67058 21618008 8 AAO51605 28829030 9 AAU95618 53854350 10 AAU95619 53854352 11 BAA97003 8777413 12 CAE76429 38567134 13 EAK90852 46431255 14 EAL32264 54643520 15 NP_015297 6325229 16 NP_568694 30695411 17 NP_572414 24640423 18 NP_596686 19113478 19 NP_851154 30695409 20 NP_908411 34894172 21 NP_974900 42573608 22 NP_974901 42573610 23 NP_984262 45188039 24 XP_389497 46134945 25 XP_401186 49074048 26 XP_405546 49087148 27 XP_449306 50293789 28 XP_449306 50293789 29 XP_450298 50899020 30 XP_453599 50307241 31 XP_460741 50424309 32 XP_500646 50546253

TABLE 8 Examples of HMG-CoA synthase polypeptides. Genbank Row ACCESSION Genbank GI 1 B55729 1083370 2 P54869 1708235 3 S13887 86312 4 S27197 284048 5 AAA37076 387072 6 AAF89580 9621905 7 AAH00297 33991031 8 AAH31363 21618633 9 AAH42929 27552834 10 AAH79694 50925193 11 AAH83543 54035469 12 AAO52569 28830079 13 AAP35966 30583443 14 BAB23657 12836439 15 BAC04559 21754758 16 BAC05233 21758044 17 CAA52032 1772495 18 CAC18553 11602786 19 CAG33131 48145817 20 CAH92111 55730782 21 CAI22408 56205097 22 EAK97451 46438115 23 EAL25034 54635631 24 NP_002121 54020720 25 NP_013580 6323509 26 NP_032282 31560689 27 NP_058964 8393538 28 NP_593859 19114771 29 NP_666054 31981842 30 NP_725570 24654139 31 NP_775117 27465521 32 NP_957379 41055180 33 NP_983739 45187516 34 NP_990742 45382279 35 NP_999545 47523816 36 XP_315872 58387870 37 XP_323241 32405256 38 XP_368218 39973655 39 XP_389442 46134253 40 XP_397202 48141273 41 XP_402977 49078452 42 XP_409060 49095198 43 XP_446972 50289085 44 XP_453529 50307101 45 XP_456470 50405663 46 XP_506052 50557288 47 XP_513693 55587844 48 XP_536483 57085299 49 XP_569805 58265298 50 XP_571930 58269548

TABLE 9 Examples of HMG-CoA reductase polypeptides. Genbank Row ACCESSION Genbank GI 1 A23586 90238 2 O74164 11132850 3 P51639 1708252 4 P54960 1708251 5 Q12649 18276268 6 Q29512 2495262 7 Q9Y7D2 11133211 8 S30338 422383 9 S72194 7450066 10 AAA36989 387052 11 AAA37077 305355 12 AAA49740 214237 13 AAD20975 9817458 14 AAH74197 49257596 15 AAL09351 15824453 16 AAO85434 29468180 17 AAP72015 32165622 18 AAR02862 45272118 19 AAT92819 51013051 20 BAC20567 23574646 21 CAA63970 4376229 22 CAE47850 41581201 23 CAF92135 47213283 24 CAH92577 55731745 25 EAK94577 46435190 26 EAL20195 50257490 27 AAF80374 8886086 28 NP_013555 6323483 29 NP_013636 6323565 30 NP_032281 56119096 31 NP_037266 40538852 32 NP_588235 19075735 33 NP_985010 45190756 34 NP_989816 45383193 35 NP_999724 47551099 36 XP_324892 32408825 37 XP_364130 39955070 38 XP_389373 46134115 39 XP_400629 49072680 40 XP_405730 49087632 41 XP_407954 49092986 42 XP_449268 50293713 43 XP_451740 50303597 44 XP_458872 50420671 45 XP_503558 50552167 46 XP_536323 57084803 47 XP_571450 58268588

TABLE 10 Examples of mevalonate kinase polypeptides. Genbank Row ACCESSION Genbank GI 1 XP_386088 46123069 2 XP_408006 49093090 3 XP_370449 39978123 4 EAL04797 46445529 5 XP_322935 32404644 6 NP_001007 55925207 7 XP_460851 50424525 8 XP_567851 58260882 9 XP_567850 58260880 10 AAQ02416 33303805 11 CAA53059 450346 12 AAH16140 16359371 13 AAH05606 13542811 14 XP_403111 49078786 15 XP_452532 50305147 16 CAG08527 47226511 17 XP_446138 50287417 18 AAO51522 28828936 19 NP_985191 45190937 20 XP_500956 50546973 21 NP_013935 6323864 22 AAD45421 5578718 23 NP_920723 37532842 24 NP_851084 30690651 25 AAL18925 16417946 26 NP_788338 28573850 27 AAU20834 51988124 28 AAU87813 52839819 29 AAU20835 51988125 30 YP_183887 57641409 31 NP_143478 14591399 32 BAA24409 2804172 33 NP_126232 14520757 34 XP_522574 55639331 35 NP_071114 11499870 36 XP_423949 50797461 37 NP_633786 21227864 38 ZP_002971 48840229 39 EAH50787 44170778 40 NP_615566 20089491 41 1VISA 40890012 42 EAK03559 44549994 43 NP_248080 15669275 44 1KKHA 20150886 45 Q50559 2497518 46 CAF88123 47200914 47 NP_275189 15678075 48 EAI88745 44383877 49 ZP_002040 46141948 50 XP_543435 57105916 51 EAI38920 44313360 52 NP_148611 14602065 53 EAD08953 43286228 54 EAD45697 43361720 55 YP_134862 55377012 56 NP_720650 24378695 57 NP_614276 20094429 58 E84270 25409931 59 NP_691146 23097680 60 ZP_003233 48870579 61 AAG02440 9937386 62 EAD12278 43292898 63 NP_498328 17555862 64 EAB31483 42928976 65 ZP_003319 50590618 66 NP_814642 29375488 67 AC1434 25514495 68 ZP_003577 53796847 69 EAD82048 43454743 70 CAE73618 39586491 71 YP_012624 46906235 72 NP_988455 45358898 73 ZP_002348 47097293 74 ZP_002862 48824993 75 ZP_002307 47093020 76 NP_597102 19173299 77 CAD24422 20429111 78 NP_785308 28378416 79 EAA39098 29247539 80 NP_819638 29653946 81 EAH49746 44168765 82 EAH49745 44168764 83 NP_378182 15922513 84 ZP_000459 23002259 85 H90181 25393827 86 YP_054120 50405028 87 BAB07790 9695270 88 AAG02435 9937379 89 NP_560495 18313828 90 YP_187834 57866187 91 EAK40782 44602942 92 CAC51370 15212070 93 AAG02424 9937364 94 YP_185521 57651465 95 YP_040044 49482820 96 YP_194037 58337452 97 D86675 25400965 98 NP_763916 27467279 99 CAF89434 47197810 100 EAF38333 43767792 101 EAK46841 44611394 102 H89827 25507776 103 ZP_003149 48861061 104 EAK17824 44570143 105 EAH86276 44235719 106 YP_118418 54024176 107 ZP_003196 48865749 108 AAG02430 9937372 109 NP_269075 15674901 110 NP_802520 28896170 111 AAL97579 19748102 112 ZP_003666 56808907 113 NP_965060 42519130 114 NP_819639 29653947 115 EAD97024 43484567 116 BAD86800 57753870

TABLE 11 Examples of phosphomevalonate kinase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAA34596 171479 2 XP_452514 50305111 3 NP_985210 45190956 4 XP_446144 50287429 5 XP_462340 50427455 6 EAL04096 46444824 7 EAL03941 46444668 8 XP_503619 50552418 9 XP_389940 46136497 10 XP_329795 32418634 11 XP_369652 39976529 12 XP_406448 49089559 13 NP_593421 19114333 14 XP_568385 58261950 15 EAL17628 50254887 16 AAL18926 16417948 17 BAD43274 51969164 18 BAD44652 51971975 19 XP_398375 49068172 20 BAD44486 51971643 21 F90479 25393214 22 YP_194039 58337454

TABLE 12 Examples of mevalonate pyrophosphate decarboxylase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAT93171 51013755 2 1FI4A 13786942 3 XP_455548 50311049 4 XP_445335 50285813 5 XP_456912 50409853 6 NP_986435 45200865 7 AAF19399 6625790 8 XP_328845 32416734 9 XP_505041 50555265 10 NP_594027 19114939 11 XP_364905 39963452 12 XP_390600 46137817 13 XP_408551 49094180 14 AAA34506 7544604 15 EAL18927 50256200 16 XP_568247 58261674 17 XP_402794 49077992 18 AAH81784 51980639 19 EAL00166 46440864 20 NP_619597 20149736 21 NP_112324 13592005 22 BAC40852 26354448 23 XP_546783 57087071 24 Q99JF5 23814095 25 AAH63907 39645379 26 CAF99534 47230341 27 AAP35576 30582699 28 AAP36301 30584105 29 AAL18927 16417950 30 AAV32433 54292590 31 AAP68208 31711704 32 AAM64988 21593039 33 NP_566995 18410026 34 XP_423130 50771155 35 AAM65192 21593243 36 NP_001007 55925435 37 NP_573068 28571205 38 BAD27942 50252009 39 T47584 11281655 40 XP_307373 31196851 41 CAE73245 39591192 42 NP_496966 17537201 43 XP_393230 48121058 44 G90479 25393662 45 NP_496967 17537203 46 NP_691147 23097681 47 EAL29282 54640164 48 AD1434 25515042 49 ZP_002308 47093021 50 YP_012625 46906236 51 ZP_002348 47097294 52 NP_819637 29653945 53 NP_376888 15921219 54 ZP_003319 50590617 55 NP_585805 19074299 56 YP_187835 57866188 57 CAD24423 20429112 58 AAG02431 9937373 59 NP_763917 27467280 60 AAG02446 9937394 61 ZP_002863 48824994 62 AAG02441 9937387 63 YP_185522 57651466 64 A89828 25505863 65 NP_814641 29375487 66 YP_040045 49482821 67 NP_785307 28378415 68 ZP_003196 48865750 69 ZP_003233 48870580 70 E86675 25400967 71 EAE31110 43552684 72 BAB07791 9695271 73 CAC51371 15212071 74 ZP_000459 23002258 75 NP_965061 42519131 76 BAD86801 57753871 77 YP_194038 58337453 78 YP_118419 54024177 79 EAK18820 44571499 80 EAI85935 44379784 81 NP_721336 24379381 82 D95044 25388338 83 AAG02456 9937408 84 C97914 25511486 85 EAK47683 44612560 86 EAB86425 43039778 87 YP_140971 55822530 88 YP_139081 55820639 89 BAD07376 40882372 90 NP_968512 42523132 91 EAI06705 44265427 92 YP_060018 50914046 93 AAG02451 9937401 94 NP_269076 15674902 95 ZP_003666 56808906 96 NP_688323 22537472 97 NP_735832 25011437 98 EAC40267 43149093 99 AAL97580 19748103 100 EAI76915 44367119 101 EAD35042 43339207 102 YP_073129 51598941 103 EAI90092 44385501 104 BAB07818 9711347 105 EAD72850 43433025 106 NP_212820 15595031 107 YP_124337 54297968 108 YP_096056 52842257 109 EAA39903 29248368 110 EAH06252 44088237 111 YP_127354 54294939 112 EAD45753 43361830 113 NP_802519 28896169

TABLE 13 Examples of IPP isomerase polypeptides. Genbank Row ACCESSION Genbank GI 1 NP_015208 6325140 2 XP_448008 50291151 3 NP_983828 45187605 4 XP_455121 50310203 5 XP_462358 50427491 6 EAL01685 46442395 7 XP_504974 50555131 8 XP_328425 32415894 9 XP_367200 39971619 10 XP_389898 46136413 11 XP_404716 49085144 12 CAD37150 21627818 13 NP_595164 19111956 14 XP_566641 58258457 15 XP_402453 49077100 16 O35586 6225528 17 AAP36609 30584713 18 AAF37873 7188790 19 NP_445991 16758306 20 O42641 6225529 21 BAA33979 3790386 22 Q13907 6225527 23 AAH22418 48257241 24 AAH19227 48257312 25 AAH57827 35505325 26 NP_004499 40018633 27 AAH89786 58477715 28 CAH91844 55730243 29 XP_418561 50732281 30 AAH06999 48257093 31 CAF98782 47225155 32 NP_808875 29366820 33 XP_507622 55633353 34 AAH82648 52139082 35 NP_001011 58332496 36 AAF29976 6856556 37 AAG10423 9971806 38 O48964 6225525 39 AAF29973 6856550 40 AAF29977 6856558 41 AAQ84167 35186998 42 AAF29974 6856552 43 Q39472 6225526 44 S49588 1085973 45 AAL91980 19568939 46 BAB40973 13603406 47 AAF29975 6856554 48 T52027 25493162 49 AAL91979 19568937 50 T46812 11362218 51 T51248 11362217 52 BAB40974 13603408 53 O48965 6225532 54 XP_225509 34877710 55 XP_506401 51963472 56 AAF29978 6856560 57 AAH76541 50369278 58 AAT94033 51038230 59 XP_225502 34876517 60 Q39471 6225533 61 AAB67743 1213450 62 NP_197148 22326844 63 BAB09611 9759126 64 AAD41766 5305669 65 AAB67741 1213442 66 XP_395125 48101420 67 AAN28784 23505849 68 AAF36996 7110585 69 BAB16690 15289752 70 AAQ14869 33340598 71 BAC65421 28971819 72 S71369 2129625 73 AAF29979 6856562 74 AAF29980 6856564 75 AAP21674 30267831 76 Q39664 6225534 77 NP_650962 24648688 78 AAM50284 21429130 79 XP_321388 58395620 80 Q9BXS1 20978506 81 T07979 7484383 82 XP_225508 34876527 83 AAT92102 51011386 84 XP_225507 34876555 85 XP_344623 34876537 86 S44843 630677 87 XP_225498 27687955 88 AAT08468 47013849 89 EAI79636 44370808 90 CAE75055 39587401 91 EAL04047 46444775 92 XP_225528 34876543 93 XP_544282 57040602 94 XP_225511 27688013 95 P26173 114853 96 EAJ04069 44405322 97 EAH27496 44127513 98 AAF91499 9653280 99 AAM48661 21328655 100 EAK17826 44570145 101 EAD59515 43391069 102 YP_128702 54307682 103 EAK66656 44639203 104 YP_118189 54023947 105 T50740 11282665 106 ZP_002077 46193541 107 EAK16470 44568229 108 YP_165403 56695056 109 EAD08775 43285885 110 YP_195623 58616494 111 EAI38918 44313358 112 NP_930583 37527239 113 YP_160254 56478665 114 EAH69842 44206571 115 EAK26254 44582307 116 AAR24381 38569721 117 AAM48607 21328600 118 EAD82049 43454744 119 ZP_001924 45914126 120 YP_056780 50843553 121 YP_050880 50121713 122 EAF29235 43749645 123 NP_630823 21225044 124 Q82MJ7 34582349 125 ZP_003374 52010110 126 AAS75819 45737905 127 Q8KP37 30913023 128 XP_507621 55633351 129 XP_344621 34876521 130 XP_346322 34880719 131 YP_152060 56414985 132 AAT42442 48429280 133 Q9KK75 13878536 134 NP_806649 29143307 135 YP_063124 50955836 136 Q8FND7 46395593 137 CAF20647 41326485 138 Q8NN99 23821718 139 Q7X5H2 46395586 140 NP_336246 15841209 141 Q83MJ9 46395588 142 P60923 46395576 143 Q8FE75 31563050 144 1R67A 38493022 145 Q9KWD1 13878537 146 Q7VEU0 46395585 147 B84333 25410326 148 NP_417365 16130791 149 E85944 25355426 150 1HZTA 15826050 151 1PVFB 50513321 152 EAD63579 43403471 153 1I9AB 13786886 154 YP_012992 46906603 155 ZP_002293 47091503 156 EAI37194 44310821 157 YP_137864 55380014 158 CAD92056 42516867 159 1OW2B 42543244

TABLE 14 Examples of FPP synthase polypeptides. Genbank Row ACCESSION Genbank GI 1 Q92250 2497455 2 XP_363065 39948036 3 XP_386960 46124813 4 Q92235 3122099 5 XP_412149 49116518 6 XP_503599 50552378 7 NP_593299 19114211 8 CAD42869 21955860 9 XP_448787 50292709 10 NP_012368 6322294 11 T42081 7433997 12 EAK93751 46434339 13 XP_451300 50302727 14 XP_571137 58267962 15 XP_460720 50424267 16 NP_984739 45190485 17 BAD15361 46367743 18 S71433 7433991 19 CAA65643 1523990 20 XP_399061 49069544 21 S71432 7433990 22 AAH68912 46249832 23 1FPS 1065289 24 P08836 3915686 25 AAH83515 53733369 26 1UBX 1942050 27 1UBY 1942051 28 AAF37872 7188788 29 NP_803463 29135293 30 AAK63847 14488053 31 AAV58896 55710092 32 T06272 7433988 33 JC4846 2117737 34 P05369 120478 35 O24241 25452945 36 O24242 25452946 37 AAH59125 37590777 38 AAH48497 28913418 39 AAP74720 32329199 40 CAG11850 47225367 41 AAM51429 21436457 42 AAP74719 32329197 43 AAM08927 20135548 44 XP_537252 57089113 45 AAQ56011 34013692 46 AAQ14872 33340604 47 AAQ14871 33340602 48 AAD17204 4324960 49 AAH87886 56789674 50 AAK68152 14573639 51 AAA52423 182399 52 S66470 2129849 53 CAA29064 4725 54 CAI12715 55957735 55 BAA03523 40788949 56 P14324 1346031 57 S66471 2129850 58 AAA35820 182405 59 CAA59170 1491641 60 BAB16687 15289750 61 CAA72793 1922251 62 CAH91070 55728661 63 AAK58594 14279425 64 AAB07264 1146159 65 Q09152 21431776 66 O64905 6016044 67 BAB60822 14422406 68 S52009 1076319 69 NP_917118 34911542 70 AAD32648 4894899 71 AAA40960 203582 72 AAR27053 38684029 73 AAU43998 52353430 74 AAL82595 18958450 75 NP_917069 34911444 76 XP_228802 34879769 77 BAD81810 56785155 78 AAN62522 24796660 79 NP_595334 19112126 80 T52066 25458583 81 AAL49067 17946048 82 CAA08919 3395483 83 XP_547662 57089869 84 EAL26135 54636732 85 BAB60821 14422404 86 AAP74721 32329201 87 XP_496902 51466663 88 XP_474182 50929309 89 CAA87327 1160178 90 BAD20729 47776234 91 BAC53873 30984142 92 BAB69490 15991313 93 NP_974565 42572937 94 CAA08918 5678609 95 AAP86267 32527731 96 AAO17735 30522953 97 AAK71861 14647139 98 AAL73357 18478919 99 AAO63552 29124957 100 CAI00471 56498227 101 NP_701155 23508486 102 XP_474180 50929305 103 AAL73358 18478922 104 EAH48995 44167328 105 NP_493027 17508563 106 CAE71711 39580204 107 XP_487220 51766977

TABLE 15 Examples of GGPP synthase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAT92871 51013155 2 XP_447025 50289191 3 NP_984623 45190369 4 XP_390273 46137163 5 XP_404791 49085320 6 XP_368486 39974191 7 Q92236 6831550 8 AAO85432 29468176 9 XP_572774 58271236 10 XP_502923 50550901 11 AAK11525 13021716 12 XP_326920 32412880 13 CAF32032 42820719 14 BAD29965 50355599 15 XP_384767 46117498 16 BAD29970 50355631 17 CAB89115 7649674 18 CAG09545 47229030 19 CAI13753 55960163 20 AAH69913 47124116 21 AAH67768 45709211 22 XP_455003 50309979 23 P56966 9296978 24 NP_001007 56090562 25 AAT65717 49409613 26 NP_956329 41053321 27 BAA90525 6899844 28 XP_405729 49087630 29 AAK11531 13021724 30 XP_412280 49119197 31 AAC05273 2944400 32 NP_523958 24660002 33 XP_402074 49076128 34 EAL30191 54641441 35 XP_536340 57084951 36 XP_424685 50811194 37 AAH06798 13905030 38 AAP06018 29841005 39 XP_460338 50423511 40 AAC05595 2957271 41 EAK92197 46432727 42 XP_535573 57108760 43 AAH83212 53734594 44 XP_486466 51827552 45 CAH18006 51469024 46 CAA75568 3549881 47 XP_397455 48143654 48 XP_410947 49101294 49 XP_381914 46109712 50 XP_364478 39959279 51 XP_360889 39942704 52 XP_369218 39975655 53 XP_406544 49089926 54 XP_367595 39972409 55 XP_363775 39952117 56 XP_368486 39974191 57 XP_390273 46137163 58 Q92236 6831550 59 AAK11525 13021716 60 CAF32032 42820719 61 XP_404791 49085320 62 AAO85432 29468176 63 BAD29965 50355599 64 BAD29970 50355631 65 BAA90525 6899844 66 AAT65717 49409613 67 XP_384767 46117498 68 CAB89115 7649674 69 XP_572774 58271236 70 AAK11531 13021724 71 XP_502923 50550901 72 CAI13753 55960163 73 CAG09545 47229030 74 XP_412280 49119197 75 P56966 9296978 76 NP_001007 56090562 77 AAH69913 47124116 78 AAH67768 45709211 79 NP_956329 41053321 80 EAL30191 54641441 81 XP_424685 50811194 82 XP_536340 57084951 83 NP_523958 24660002 84 AAC05273 2944400 85 XP_405729 49087630 86 AAC05595 2957271 87 XP_402074 49076128 88 AAP06018 29841005 89 AAH06798 13905030 90 XP_535573 57108760 91 AAH83212 53734594 92 AAP21085 30097620 93 NP_984623 45190369 94 XP_447025 50289191 95 AAT92871 51013155 96 XP_486466 51827552 97 XP_410947 49101294 98 XP_397455 48143654 99 XP_455003 50309979 100 EAK92197 46432727 101 XP_381914 46109712 102 XP_460338 50423511 103 CAH18006 51469024 104 XP_360889 39942704 105 XP_406544 49089926 106 XP_364478 39959279 107 XP_363775 39952117 108 XP_367595 39972409 109 XP_369218 39975655 110 C39273 483124 111 BAB79600 18143445 112 BAA14124 216682 113 AAN85596 27228290 114 AAA32797 413730 115 Q08291 585326 116 S52584 1073293 117 S53722 1076576 118 AAC44848 1842242 119 BAA19583 1944371 120 S71230 2129674 121 BAA23157 2578822 122 AAC77874 3885426 123 CAB38744 4490594 124 BAA78047 4958920 125 BAA82613 5631295 126 CAB56064 5912297 127 BAA86284 6277254 128 T11021 7447356 129 AAF78199 8650415 130 AAG10424 9971808 131 CAC10561 10637876 132 T50879 11279298 133 BAB01343 11994221 134 Q42698 13431546 135 Q43133 13431547 136 P54976 13878921 137 BAB50600 14023995 138 BAB60678 14325238 139 BAB60820 14422402 140 NP_189589 15228704 141 NP_188651 15231055 142 NP_188069 15231869 143 NP_188073 15231881 144 AAL01997 15553715 145 AAL01998 15553717 146 NP_252732 15599238 147 NP_245470 15602398 148 NP_390308 16079484 149 NP_440010 16329282 150 NP_440010 16329282 151 AAL17614 17352451 152 NP_520343 17546941 153 AAL76349 18645048 154 AAM21638 20386366 155 AAM21639 20386368 156 NP_622916 20807745 157 AAM48650 21328644 158 NP_659794 21492720 159 AAM64496 21592547 160 AAM65107 21593158 161 NP_680811 22297564 162 ZP_000474 23003800 163 ZP_001252 23469933 164 NP_698760 23502633 165 E84566 25313373 166 F85434 25313385 167 AC1245 25313389 168 E83997 25313393 169 G84566 25313395 170 AH2910 25315863 171 D87505 25398795 172 A89932 25505949 173 F97685 25520741 174 AI3285 25527013 175 BAC42571 26450928 176 NP_785195 28378303 177 NP_790546 28867927 178 AAO63392 28950937 179 AAO93113 29893480 180 NP_833891 30022260 181 AAP59037 31621279 182 ZP_001374 32039216 183 NP_864766 32471772 184 NP_875521 33240579 185 NP_881399 33593755 186 NP_884694 33597051 187 NP_888456 33600896 188 NP_893187 33861626 189 NP_894940 33863380 190 NP_896835 33865276 191 NP_896835 33865276 192 AAQ65086 34365549 193 NP_945877 39933601 194 NP_946867 39934591 195 NP_952815 39996864 196 AAR37805 40062934 197 AAR37858 40062988 198 AAR98495 41018904 199 AAR99082 41059107 200 NP_965349 42519419 201 NP_980544 42783297 202 EAA96348 42858148 203 EAB36506 42939031 204 EAB36642 42939300 205 EAC39208 43146996 206 EAD26007 43320598 207 EAE43084 43576643 208 EAE70061 43630884 209 EAF70308 43832107 210 EAG88494 44055952 211 EAH52060 44173220 212 EAH78354 44221788 213 EAH84117 44231960 214 EAI11762 44272832 215 EAI49391 44328289 216 EAI54846 44336042 217 EAI68356 44355138 218 EAI68713 44355672 219 EAI69401 44356609 220 EAI73873 44362658 221 EAJ73634 44506168 222 EAJ77351 44511694 223 EAK70639 44644254 224 ZP_001751 45523854 225 AAS76253 45752710 226 ZP_001957 45916757 227 1RTRB 46015556 228 ZP_001863 46105954 229 ZP_002002 46107045 230 ZP_001711 46132567 231 ZP_002073 46192680 232 ZP_002074 46192861 233 AAS82860 46241274 234 ZP_002108 46308696 235 YP_010568 46579760 236 BAD18313 47076770 237 ZP_002315 47093750 238 ZP_002335 47095946 239 AAT35222 47531118 240 ZP_002401 47569437 241 ZP_002435 47573473 242 ZP_002626 48728941 243 ZP_002702 48765678 244 ZP_002705 48766028 245 ZP_002732 48768894 246 ZP_002914 48834438 247 ZP_003024 48848203 248 ZP_003129 48858958 249 ZP_003177 48863841 250 ZP_003225 48869790 251 AAT51323 49086036 252 ZP_003301 49236117 253 YP_034222 49476181 254 YP_040995 49483771 255 YP_043579 49486358 256 AAT71982 50253560 257 AAT90315 50952782 258 YP_066435 51246551 259 YP_075673 51892982 260 YP_085511 52141318 261 YP_092166 52786337 262 ZP_001298 53691368 263 YP_105136 53716444 264 YP_111769 53722784 265 ZP_003630 54030933 266 YP_129021 54308001 267 AAV74395 56122554 268 AAV74396 56122556 269 YP_148246 56420928 270 YP_156518 56461237 271 YP_162590 56551751 272 YP_171470 56750769 273 YP_175959 56964228 274 YP_186407 57650478 275 YP_190690 58038726 276 AAW66658 58201026 277 YP_194187 58337602 278 YP_197469 58579257 279 YP_201938 58582922 280 YP_196510 58617311

TABLE 16 Examples of squalene synthase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAA34597 171481 2 CAA42583 3686 3 Q9HGZ6 51704336 4 BAB12207 9955387 5 XP_453457 50306959 6 Q752X9 51701405 7 O74165 51701378 8 XP_458579 50420093 9 EAK95451 46436082 10 P78589 2499979 11 Q9Y753 51701459 12 XP_407513 49092104 13 XP_364394 39958237 14 Q7S4Z6 51701416 15 CAD60581 27764301 16 XP_389557 46135731 17 NP_595363 19112155 18 B48057 477750 19 NP_034321 34328173 20 CAH92517 55731622 21 AAF00038 6002565 22 P53798 1706773 23 NP_004453 31542632 24 AAP36671 30584837 25 1EZFC 11514497 26 AAH09251 14328083 27 AAH84016 54035372 28 I52090 2136196 29 XP_420043 50745256 30 AAH81810 51858605 31 CAE48363 50841455 32 XP_569783 58265254 33 XP_569782 58265252 34 XP_534557 57105080 35 XP_401989 49075920

TABLE 17 Examples of phytoene dehydrogenase polypeptides. Genbank Row ACCESSION 1 1613414B 2 1613414F 3 1904206A 4 2121278A 5 A86203 6 A96612 7 A99470 8 AAA24820 9 AAA34001 10 AAA50313 11 AAA64981 12 AAA91161 13 AAA99519 14 AAC44798 15 AAC44850 16 AAC48983 17 AAF78201 18 AAG10426 19 AAG14399 20 AAG28700 21 AAG50743 22 AAH85048 23 AAK51545 24 AAK51557 25 AAK64299 26 AAL02000 27 AAL15300 28 AAL38046 29 AAL73986 30 AAL80005 31 AAL91366 32 AAM45380 33 AAM48646 34 AAM63349 35 AAM94364 36 AAN75037 37 AAN85599 38 AAO24235 39 AAO46892 40 AAO46894 41 AAO53257 42 AAO53258 43 AAO64750 44 AAO93135 45 AAP59036 46 AAP79175 47 AAQ04224 48 AAQ04225 49 AAQ65246 50 AAQ65246 51 AAQ88931 52 AAR37797 53 AAR37802 54 AAR37850 55 AAR37855 56 AAR86105 57 AAR98491 58 AAR98494 59 AAR98733 60 AAS17750 61 AAT01639 62 AAT35222 63 AAT74579 64 AAT74580 65 AAT76050 66 AAT76434 67 AAT90316 68 AAU34019 69 AAW23161 70 AB2035 71 AB2064 72 AC2446 73 AF1557 74 AF2029 75 AG2103 76 AG2509 77 AH1199 78 AI2185 79 AI2273 80 B55548 81 B84327 82 B90061 83 BAA14127 84 BAA20276 85 BAA76534 86 BAB10768 87 BAB50520 88 BAB51896 89 BAB68552 90 BAB79603 91 BAB82461 92 BAB82462 93 BAB98016 94 BAC75676 95 BAC77668 96 BAC77671 97 BAD07279 98 BAD07280 99 BAD07287 100 BAD07288 101 CAA52098 102 CAA60479 103 CAA66626 104 CAB38739 105 CAB38743 106 CAB40843 107 CAB56041 108 CAB56062 109 CAB59726 110 CAB65434 111 CAB94794 112 CAC85667 113 CAD19989 114 CAD27442 115 CAD55814 116 CAE00192 117 CAE83576 118 CAF19330 119 CAF21094 120 CAF21337 121 CAH91165 122 E90061 123 EAA90383 124 EAA98598 125 EAB09790 126 EAB14136 127 EAB18725 128 EAB29729 129 EAB30992 130 EAB41377 131 EAB54727 132 EAB76679 133 EAB87028 134 EAB92587 135 EAB94459 136 EAB96864 137 EAC01884 138 EAC38895 139 EAC60360 140 EAD05874 141 EAD05999 142 EAD20520 143 EAE06978 144 EAE70773 145 EAF04985 146 EAF51354 147 EAF62819 148 EAF75453 149 EAG09111 150 EAG19412 151 EAG23070 152 EAG25053 153 EAG25054 154 EAG29279 155 EAG39845 156 EAG56100 157 EAG63013 158 EAG68633 159 EAG71574 160 EAG89835 161 EAH04928 162 EAH04936 163 EAH08586 164 EAH22597 165 EAH22853 166 EAH31648 167 EAH55579 168 EAH68071 169 EAH73302 170 EAH79041 171 EAH86965 172 EAH97108 173 EAH99977 174 EAI01660 175 EAI03576 176 EAI06784 177 EAI11087 178 EAI15261 179 EAI15547 180 EAI17521 181 EAI21398 182 EAI29728 183 EAI38468 184 EAI43591 185 EAI51589 186 EAI58453 187 EAI72974 188 EAI77885 189 EAI78272 190 EAI80262 191 EAI83937 192 EAI86664 193 EAJ00517 194 EAJ05570 195 EAJ08238 196 EAJ15524 197 EAJ18144 198 EAJ20649 199 EAJ21683 200 EAJ24413 201 EAJ28774 202 EAJ30522 203 EAJ35157 204 EAJ37407 205 EAJ39929 206 EAJ54356 207 EAJ54959 208 EAJ56207 209 EAJ58447 210 EAJ59958 211 EAJ63347 212 EAJ66054 213 EAJ67637 214 EAJ69812 215 EAJ74441 216 EAJ76472 217 EAJ76473 218 EAJ80355 219 EAJ80839 220 EAJ81408 221 EAJ86174 222 EAJ87600 223 EAJ88203 224 EAJ88682 225 EAJ92341 226 EAJ94774 227 EAJ97555 228 EAJ97958 229 EAK07654 230 EAK08513 231 EAK08529 232 EAK10609 233 EAK10614 234 EAK12902 235 EAK13034 236 EAK15092 237 EAK22483 238 EAK23222 239 EAK24187 240 EAK24674 241 EAK28785 242 EAK34731 243 EAK34742 244 EAK36883 245 EAK37522 246 EAK42705 247 EAK43213 248 EAK52580 249 EAK53452 250 EAK58759 251 EAK62665 252 EAK63558 253 F84187 254 F90272 255 G87635 256 G90413 257 H83880 258 H84320 259 JC7723 260 NP_060220 261 NP_080435 262 NP_193157 263 NP_214383 264 NP_276913 265 NP_293819 266 NP_294534 267 NP_294585 268 NP_295972 269 NP_338490 270 NP_376437 271 NP_377056 272 NP_388895 273 NP_441167 274 NP_441254 275 NP_442491 276 NP_442727 277 NP_562475 278 NP_568712 279 NP_601630 280 NP_601630 281 NP_616426 282 NP_624522 283 NP_626360 284 NP_630834 285 NP_643053 286 NP_647302 287 NP_659552 288 NP_661086 289 NP_661546 290 NP_661701 291 NP_662300 292 NP_681023 293 NP_681127 294 NP_682351 295 NP_693380 296 NP_693382 297 NP_737250 298 NP_763380 299 NP_786524 300 NP_822198 301 NP_822828 302 NP_827278 303 NP_851528 304 NP_857496 305 NP_868798 306 NP_869339 307 NP_870237 308 NP_874530 309 NP_874561 310 NP_874977 311 NP_892236 312 NP_892265 313 NP_892458 314 NP_893232 315 NP_894882 316 NP_895385 317 NP_895793 318 NP_895829 319 NP_896854 320 NP_896994 321 NP_898304 322 NP_898346 323 NP_902647 324 NP_923340 325 NP_923639 326 NP_923813 327 NP_925079 328 NP_931515 329 NP_936379 330 NP_940208 331 NP_945754 332 NP_946860 333 NP_946866 334 NP_948851 335 NP_962004 336 NP_968600 337 NP_974222 338 NP_974545 339 O49901 340 P17059 341 P54971 342 P54978 343 P54979 344 P54981 345 P54982 346 P74306 347 Q01671 348 Q02861 349 Q38893 350 Q40406 351 Q9FV46 352 Q9SE20 353 Q9SMJ3 354 Q9ZTN9 355 Q9ZTP4 356 S29314 357 S32171 358 S49624 359 S52586 360 S65060 361 T10701 362 T31463 363 T46822 364 T48646 365 T50745 366 T50749 367 T50893 368 T50910 369 T51119 370 T51123 371 XP_324732 372 XP_383241 373 XP_401825 374 XP_470568 375 XP_473486 376 XP_477063 377 XP_525801 378 XP_540198 379 YP_006049 380 YP_013621 381 YP_024310 382 YP_041986 383 YP_041988 384 YP_044561 385 YP_044564 386 YP_062471 387 YP_117947 388 YP_120612 389 YP_135077 390 YP_136483 391 YP_145331 392 YP_145348 393 YP_171014 394 YP_172823 395 YP_173078 396 YP_173207 397 YP_184572 398 YP_187368 399 YP_187371 400 YP_187371 401 YP_187371 402 ZP_000490 403 ZP_000509 404 ZP_000518 405 ZP_000566 406 ZP_000627 407 ZP_000627 408 ZP_001073 409 ZP_001081 410 ZP_001091 411 ZP_001116 412 ZP_001117 413 ZP_001119 414 ZP_001124 415 ZP_001510 416 ZP_001591 417 ZP_001593 418 ZP_001602 419 ZP_001614 420 ZP_001645 421 ZP_001650 422 ZP_001722 423 ZP_001746 424 ZP_001752 425 ZP_001770 426 ZP_001777 427 ZP_001787 428 ZP_001837 429 ZP_001867 430 ZP_002073 431 ZP_002077 432 ZP_002339 433 ZP_002680 434 ZP_002705 435 ZP_002771 436 ZP_002892 437 ZP_002916 438 ZP_002963 439 ZP_003022 440 ZP_003036 441 ZP_003107 442 ZP_003202 443 ZP_003258 444 ZP_003268 445 ZP_003269 446 ZP_003276 447 ZP_003283 448 ZP_003557 449 ZP_003559 450 ZP_003565 451 ZP_003577 452 ZP_003593 453 ZP_003595 441 ZP_003685

TABLE 18 Examples of phytoene synthase and lycopene cyclase polypeptides. Genbank Row Accession Genbank GI 1 1613414C 227040 2 A49558 1076590 3 AAA19428 506623 4 AAA32836 413732 5 AAA64982 148413 6 AAB87738 29893495 7 AAC44849 1842243 8 AAD38051 13542332 9 AAF78202 8650418 10 AAF82616 9081847 11 AAG10427 9971814 12 AAG28701 11066678 13 AAK07734 18476085 14 AAK07735 18476089 15 AAK15621 13195243 16 AAL02001 15553721 17 AAL76346 18645045 18 AAL82578 21326700 19 AAM45379 21360353 20 AAM48647 21328641 21 AAM62787 21553694 22 AAM94363 22296799 23 AAN85600 27228294 24 AAO24767 27903500 25 AAO39835 28403302 26 AAO46895 37729028 27 AAO47570 33465823 28 AAO73816 33465821 29 AAP22038 30349414 30 AAP55451 32350232 31 AAP55453 32350236 32 AAP55461 32350252 33 AAP55471 32350272 34 AAP55484 32350298 35 AAP55486 32350302 36 AAP56083 32349564 37 AAP56124 32349646 38 AAP56127 32349652 39 AAP56136 32349670 40 AAP56148 32349694 41 AAP56155 32349708 42 AAP56156 32349710 43 AAP56157 32349712 44 AAP56158 32349714 45 AAP79176 32307542 46 AAQ91837 37499616 47 AAR08445 38037628 48 AAR31885 39842609 49 AAR37803 40062932 50 AAR37856 40062986 51 AAR86104 40456029 52 AAR87868 40557193 53 AAR98492 41018901 54 AAS02284 41394357 55 AAS17009 42491736 56 AAS18307 42521626 57 AAT28184 47498515 58 AAT35222 47531118 59 AAT38473 47779181 60 AAT46069 48686711 61 AAT74581 50313418 62 AAT90319 50952786 63 AAV74394 56122551 64 AAW23162 56698928 65 AC2035 25366683 66 AC2035 25366683 67 BAB18514 11344507 68 BAB79604 18143449 69 BAD07278 40809739 70 BAD07286 40809755 71 BAD62106 54291340 72 BAD62107 54291341 73 C90061 25506636 74 CAA47625 19347 75 CAA68575 19341 76 CAB07958 1934837 77 CAB38740 4490590 78 CAB51949 5690074 79 CAB56063 5912296 80 CAB86388 7453011 81 CAB93661 8250520 82 CAB94795 8574392 83 CAC19567 11990226 84 CAC27383 12584564 85 CAD19988 18307500 86 CAD29284 57282088 87 CAE76609 38567321 88 E37802 95606 89 E84320 25410251 90 EAA98758 42863045 91 EAB01965 42869439 92 EAB04170 42873822 93 EAB07138 42879858 94 EAB09791 42885235 95 EAB19826 42905452 96 EAB35029 42936011 97 EAB41375 42948740 98 EAB78706 43024004 99 EAB92586 43052355 100 EAC06949 43081493 101 EAC18360 43104624 102 EAC25793 43119723 103 EAC29883 43128092 104 EAC32813 43133973 105 EAC33105 43134560 106 EAC38486 43145552 107 EAC52233 43173313 108 EAC60029 43189028 109 EAC68026 43204953 110 EAC96197 43261031 111 EAD08701 43285745 112 EAD20866 43310220 113 EAD32755 43334458 114 EAD38008 43345761 115 EAD50152 43370658 116 EAD50402 43371147 117 EAD81123 43452903 118 EAD93882 43478303 119 EAE12860 43516265 120 EAE16121 43522884 121 EAE31084 43552634 122 EAE35665 43561764 123 EAE44717 43579862 124 EAE46627 43583580 125 EAE47846 43586023 126 EAE72264 43635190 127 EAE76009 43642573 128 EAE86335 43662748 129 EAE89581 43669163 130 EAF18881 43728007 131 EAF64277 43819669 132 EAF67931 43827263 133 EAF84745 43861327 134 EAF94004 43880040 135 EAG06083 43903395 136 EAG21950 43933540 137 EAG43625 43973477 138 EAG50171 43985555 139 EAG57517 43999205 140 EAG62787 44009110 141 EAG65580 44014171 142 EAG68110 44018715 143 EAG72283 44026322 144 EAG78750 44037938 145 EAG80445 44041116 146 EAG93220 44064453 147 EAH04927 44085694 148 EAH08972 44093217 149 EAH10377 44095788 150 EAH22151 44117864 151 EAH31461 44134654 152 EAH50033 44169323 153 EAH64480 44196848 154 EAH79040 44223009 155 EAH99976 44255671 156 EAI02786 44259828 157 EAI02787 44259829 158 EAI03575 44260943 159 EAI05900 44264266 160 EAI61004 44344824 161 EAI70669 44358327 162 EAI83938 44377067 163 EAJ05110 44406802 164 EAJ05569 44407471 165 EAJ08876 44412338 166 EAJ35156 44449986 167 EAJ38900 44455130 168 EAJ49645 44470504 169 EAJ54357 44477026 170 EAJ60475 44485647 171 EAJ64125 44492007 172 EAJ67499 44497025 173 EAJ76471 44510405 174 EAJ76950 44511114 175 EAJ78637 44513596 176 EAJ78787 44513824 177 EAJ79616 44515082 178 EAJ80356 44516200 179 EAJ81914 44518489 180 EAJ87417 44526623 181 EAK08514 44557109 182 EAK08523 44557119 183 EAK12901 44563097 184 EAK22180 44576315 185 EAK24859 44580262 186 EAK28345 44585276 187 EAK34732 44594324 188 EAK34736 44594329 189 EAK37296 44597942 190 EAK37521 44598256 191 EAK56335 44624430 192 G84363 25410528 193 NP_274195 15677043 194 NP_284085 15794263 195 NP_294586 15805888 196 NP_388961 16078144 197 NP_441168 16330440 198 NP_443763 16519643 199 NP_624523 21218744 200 NP_630832 21225053 201 NP_662273 21674208 202 NP_682350 22299103 203 NP_693381 23099915 204 NP_786525 28379633 205 NP_822199 29827565 206 NP_822829 29828195 207 NP_851527 30795077 208 NP_868799 32475805 209 NP_874560 33239618 210 NP_879992 33592348 211 NP_884101 33596458 212 NP_889809 33602249 213 NP_892264 33860703 214 NP_895828 33864268 215 NP_898345 33866786 216 NP_902648 34498433 217 NP_902649 34498434 218 NP_924690 37521313 219 NP_931516 37528171 220 NP_946861 39934585 221 NP_949079 39936803 222 NP_962005 41409169 223 NP_968601 42523221 224 O07333 3913360 225 P08196 585746 226 P21683 30923192 227 P37269 585009 228 P37271 27735222 229 P37272 585749 230 P53797 1709885 231 P54975 1706137 232 P54977 1706139 233 P65860 54041032 234 Q9SSU8 8928282 235 Q9UUQ6 34922667 236 S22474 7489041 237 S32170 321671 238 S52587 1073300 239 S56668 2129505 240 S68307 2130144 241 T10702 7484346 242 T46594 11291807 243 T50746 11356347 244 T50895 11291816 245 XP_324765 32408567 246 XP_383242 46114448 247 XP_403902 49080862 248 YP_006040 46255128 249 YP_103126 53723680 250 YP_112342 53723357 251 YP_117945 54023703 252 YP_120611 54026369 253 YP_136628 55378778 254 YP_136629 55378779 255 YP_145340 55978284 256 YP_145343 55978287 257 YP_160917 56479328 258 YP_160918 56479329 259 YP_162605 56551766 260 YP_172822 56752121 261 YP_187369 57652299 262 YP_192648 58040684 263 ZP_000044 22956752 264 ZP_001091 53688068 265 ZP_001591 53763709 266 ZP_001657 45514234 267 ZP_001690 46132223 268 ZP_001746 45523280 269 ZP_001837 53771530 270 ZP_001867 45546711 271 ZP_002096 46204978 272 ZP_002248 46324513 273 ZP_002450 47575031 274 ZP_002680 48763469 275 ZP_002710 48766450 276 ZP_002791 48782680 277 ZP_002892 48832182 278 ZP_002916 48834623 279 ZP_003036 48849426 280 ZP_003269 48893702 281 ZP_003351 52007802 282 ZP_003487 53730362 283 ZP_003501 53759405 284 ZP_003591 53798896 285 ZP_003628 54030691

TABLE 19 Examples of carotenoid ketolase polypeptides. Accession Row Number GI Number 1 AAA99932 609575 2 AAB48668 1870215 3 AAC25611 2541936 4 AAF78203 8650419 5 AAH16427 16741158 6 AAN03484 22597194 7 AAN85497 26541510 8 AAN86030 33439708 9 AAO64399 28976134 10 AAQ23139 33621091 11 AAT35222 47531118 12 AAT35555 47558911 13 AAV41372 55139370 14 AB2307 25530134 15 AF2204 25533132 16 BAB54999 14028447 17 BAB58879 14270087 18 BAC98366 37360914 19 CAA60478 2654318 20 CAB56059 5912292 21 D87673 25398945 22 EAA79304 42823978 23 EAA80363 42826055 24 EAA81403 42828115 25 EAA84711 42834481 26 EAB82380 43031476 27 EAB86624 43040184 28 EAC05755 43079085 29 EAD12219 43292778 30 EAD71182 43427899 31 EAD94927 43480380 32 EAF11582 43712986 33 EAF98072 43888329 34 EAG19345 43928738 35 EAG38273 43963688 36 EAG79800 44039853 37 EAG96474 44070318 38 EAH00349 44077315 39 EAH36448 44143633 40 EAH40683 44151265 41 EAH53180 44175316 42 EAH96648 44250729 43 EAI05260 44263397 44 EAI17468 44281329 45 EAI53009 44333409 46 EAI54054 44334878 47 EAI67818 44354362 48 EAI68153 44354875 49 EAI89684 44384943 50 EAJ27674 44439188 51 EAJ45589 44464684 52 EAJ45589 44464684 53 EAJ67118 44496466 54 EAJ74221 44507022 55 EAJ74653 44507662 56 EAJ88396 44528064 57 EAJ88887 44528792 58 EAK06069 44553531 59 EAK11467 44561166 60 EAK16824 44568733 61 EAK28828 44585942 62 EAK28828 44585942 63 EAK31112 44589271 64 EAK42591 44605441 65 NP_045063 11465545 66 NP_081575 27754029 67 NP_338204 15843167 68 NP_440788 16330060 69 NP_441220 16330492 70 NP_682690 22299443 71 NP_770721 27379192 72 NP_848964 30468077 73 NP_857223 31794730 74 NP_881760 33594116 75 NP_882469 33594826 76 NP_886657 33599097 77 NP_895643 33864083 78 NP_896386 33864827 79 NP_897461 33865902 80 NP_924674 37521297 81 NP_927525 37524181 82 NP_947075 39934799 83 P54972 1706150 84 Q39982 2498257 85 Q44261 2498256 86 T31123 11361063 87 XP_330780 32420673 88 XP_368852 39974923 89 XP_380194 46102628 90 XP_383758 46115480 91 XP_405100 49086048 92 XP_409222 49095522 93 YP_102417 53725671 94 YP_108945 53719959 95 YP_132414 54302421 96 YP_154670 56459389 97 YP_166682 56696325 98 YP_168846 56698471 99 YP_172377 56751676 100 ZP_001068 23124870 101 ZP_001112 53688676 102 ZP_001607 53764743 103 ZP_001757 46118877 104 ZP_001787 53736018 105 ZP_002218 46321435 106 ZP_002456 47575608 107 ZP_003028 48848557 108 ZP_003107 48856640 109 ZP_003264 48893204 110 ZP_003458 53688805 111 ZP_003513 53763576

TABLE 20 Examples of carotenoid hydroxylase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAC44852 1842246 2 AAC49443 1575296 3 AAD54243 5852870 4 AAG10430 9971820 5 AAG10793 9988836 6 AAG33636 11245486 7 AAL80006 19071768 8 AAM44971 21280903 9 AAM51300 21436107 10 AAM77007 21734857 11 AAN85601 27228295 12 AAO53295 28911949 13 AAS48097 44887642 14 AAS55552 45184599 15 AAS88426 46326968 16 AAT48741 49036137 17 AAT84408 50844570 18 AAV85452 56267980 19 AAV85453 56267982 20 BAA14129 216687 21 BAB79605 18143450 22 BAC77670 31790567 23 BAD07283 40809749 24 BAD07291 40809765 25 CAA70427 2956671 26 CAA70888 2956717 27 CAB55625 5870598 28 CAB55626 5870600 29 CAB56060 5912293 30 CAC06712 9968545 31 CAC95130 33145986 32 EAB30128 42926157 33 EAC49462 43167766 34 EAC86129 43241003 35 EAD61089 43395962 36 EAD76156 43443111 37 EAD88640 43467793 38 EAE27903 43546376 39 EAE28203 43546980 40 EAE78743 43647896 41 EAF12173 43714211 42 EAH29370 44130906 43 EAH44202 44158360 44 EAI00766 44256844 45 EAI29017 44298625 46 EAJ30844 44443849 47 EAJ72524 44504516 48 EAK10611 44559981 49 EAK53455 44620561 50 EAK63955 44635271 51 H90469 25394049 52 NP_745389 26989964 53 NP_922503 37536402 54 P54973 1706152 55 Q44262 2498258 56 S52982 1073291 57 XP_473611 50928167 58 YP_024309 48478603 59 ZP_003055 48851297 60 ZP_003107 48856620

TABLE 21 Examples of astaxanthin synthase polypeptides and putative astaxanthin synthase polypeptides. Genbank Row ACCESSION Genbank GI 1 AAM56288 21501451 2 XP_571276 58268240 3 EAL20013 50257304 4 XP_401804 49075484 5 XP_397817 49067054 6 XP_399595 49070612 7 XP_403279 49079218 8 XP_382294 46110473 9 XP_406021 49088382 10 XP_381224 46108332 11 XP_391479 46139577 12 XP_569261 58264210 13 EAL22841 50260180 14 XP_359866 39940658

TABLE 22 Examples of carotenoid epsilon hydroxylase polypeptides. ACCESSION GI PROTEIN DESCRIPTION ABB52076 79155148 putative epsilon-ring carotene hydroxylase [Daucus carota subsp. sativus] BAD94136 62319017 Cytochrom P450-like protein [Arabidopsis thaliana] ABD28565 87162770 E-class P450, group I [Medicago truncatula] AAT28222 47498772 putative 97B2-like cytochrome P450 [Ginkgo biloba] ABC68396 85001685 cytochrome P450 monooxygenase CYP97A [Glycine max] ABC59110 84514203 cytochrome P450 monooxygenase CYP97B [Medicago truncatula] NP_190881 42565881 LUT1 (LUTEIN DEFICIENT 1); oxygen binding [Arabidopsis thaliana] ABB47954 78708979 cytochrome P450 monooxygenase, putative [Oryza sativa (japonica cultivar-group)] NP_922604 37536604 putative cytochrome P450 monooxygenase [Oryza sativa (japonica cultivar-group)]

TABLE 23 Examples of lycopene cyclase polypeptides, beta and epsilon subunits. ACCESSION GI PROTEIN DESCRIPTION AAK07431 12746307 lycopene epsilon-cyclase [Adonis palaestina] ABB52073 79154988 putative lycopene epsilon cyclase [Daucus carota subsp. sativus] Q38932 27735211 Lycopene epsilon cyclase, chloroplast precursor AAB53336 1399181 lycopene epsilon cyclase AAG10428 9971816 epsilon cyclase [Tagetes erecta] AAK07434 12746313 lycopene epsilon-cyclase [Lactuca sativa] AAM45382 21360359 epsilon cyclase [Tagetes erecta] O65837 11132841 Lycopene epsilon cyclase, chloroplast precursor AAL69394 18419661 lycopene epsilon-cyclase [Spinacia oleracea] BAE79549 87299433 lycopene epsilon-cyclase [Chrysanthemum × morifolium] XP_463351 50901836 putative lycopene epsilon-cyclase [Oryza sativa (japonica cultivar-group)] AAS48096 44887640 epsilon lycopene cyclase [Citrus sinensis] AAX92679 62638188 lycopene epsilon cyclase [Citrus maxima] AAL92114 19569601 lycopene epsilon-cyclase [Citrus × paradisi] AAK07433 12746311 lycopene epsilon-cyclase [Solanum tuberosum] AAL47019 17864021 lycopene epsilon-cyclase [Citrus sinensis] AAT46065 48686703 chloroplast lycopene epsilon-cyclase precursor [Chlamydomonas reinhardtii] BAD07293 40809769 lycopene epsilon-cyclase [Citrus limon] BAD07285 40809753 lycopene epsilon-cyclase [Citrus sinensis] BAD07277 40809737 lycopene epsilon-cyclase [Citrus unshiu] EAJ62839 44489138 unknown [environmental sequence] BAE43547 73993068 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43550 73993074 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43557 73993088 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43558 73993090 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43553 73993080 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43545 73993064 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43556 73993086 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43552 73993078 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43560 73993094 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43554 73993082 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43551 73993076 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43519 73993012 putative lycopene beta cyclase [Cryptomeria japonica] BAE43535 73993044 putative lycopene beta cyclase [Cryptomeria japonica] BAE43541 73993056 putative lycopene beta cyclase [Cryptomeria japonica] BAE43542 73993058 putative lycopene beta cyclase [Cryptomeria japonica] BAE43517 73993008 putative lycopene beta cyclase [Cryptomeria japonica] BAE43534 73993042 putative lycopene beta cyclase [Cryptomeria japonica] BAE43537 73993048 putative lycopene beta cyclase [Cryptomeria japonica] BAE43533 73993040 putative lycopene beta cyclase [Cryptomeria japonica] BAD02774 38603277 putative lycopene beta cyclase [Cryptomeria japonica] BAD02766 38603261 putative lycopene beta cyclase [Cryptomeria japonica] BAE43540 73993054 putative lycopene beta cyclase [Cryptomeria japonica] BAE43514 73993002 putative lycopene beta cyclase [Cryptomeria japonica] BAE43544 73993062 putative lycopene beta cyclase [Cryptomeria japonica] BAE43538 73993050 putative lycopene beta cyclase [Cryptomeria japonica] BAE43528 73993030 putative lycopene beta cyclase [Cryptomeria japonica] BAE43546 73993066 putative lycopene beta cyclase [Taxodium distichum var. distichum] BAE43526 73993026 putative lycopene beta cyclase [Cryptomeria japonica] BAE43543 73993060 putative lycopene beta cyclase [Cryptomeria japonica] BAD02742 38603213 putative lycopene beta cyclase [Cryptomeria japonica] BAD02770 38603269 putative lycopene beta cyclase [Cryptomeria japonica] BAE43522 73993018 putative lycopene beta cyclase [Cryptomeria japonica] BAE43559 73993092 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43527 73993028 putative lycopene beta cyclase [Cryptomeria japonica] BAE43548 73993070 putative lycopene beta cyclase [Taxodium distichum var. distichum] AAF44700 14550425 lycopene beta-cyclase [Citrus sinensis] BAE43555 73993084 putative lycopene beta cyclase [Taxodium distichum var. imbricarium] BAE43549 73993072 putative lycopene beta cyclase [Taxodium distichum var. distichum] AAU14144 51922063 lycopene beta-cyclase [Citrus sinensis] AAN86060 27261727 lycopene cyclase [Citrus unshiu] AAR89632 40756518 lycopene-beta-cyclase [Citrus maxima] AAM21152 20530862 lycopene beta-cyclase [Citrus sinensis] AAD38049 13959731 lycopene cyclase [Citrus × paradisi] AAU05146 51511939 lycopene beta-cyclase [Citrus sinensis] AAU05145 51511937 lycopene beta-cyclase [Citrus sinensis] AAK07430 12746305 lycopene beta-cyclase [Adonis palaestina] ABB72443 82394885 lycopene beta-cyclase [Citrus sinensis] BAE79544 87299423 lycopene beta-cyclase [Chrysanthemum × morifolium] BAE78471 85717882 lycopene beta cyclase [Taraxacum officinale] Q43415 11133019 Lycopene beta cyclase, chloroplast precursor AAF23013 6665782 lycopene epsilon-cyclase [Daucus carota] ABB52071 79154899 putative lycopene beta cyclase [Daucus carota subsp. sativus] AAW88382 59665024 lycopene beta-cyclase [Lycium barbarum] AAG10429 9971818 beta cyclase [Tagetes erecta] AAM45381 21360357 beta cyclase [Tagetes erecta] AAM14335 20259239 putative lycopene beta cyclase [Arabidopsis thaliana] AAO18661 27728515 lycopene beta-cyclase [Zea mays] AAA81880 735882 lycopene cyclase Q43503 11133022 Lycopene beta cyclase, chloroplast precursor S66350 2129931 lycopene beta-cyclase (EC 5.5.1.—) - tomato XP_464409 50905841 putative lycopene beta-cyclase [Oryza sativa (japonica cultivar-group)] CAD70565 45237491 lycopene cyclase [Bixa orellana] Q43578 11133025 Lycopene beta cyclase, chloroplast precursor AAL92175 19569782 beta-lycopene cyclase [Sandersonia aurantiaca] AAX54906 61742130 putative chloroplast lycopene beta cyclase precursor [Chlamydomonas reinhardtii] S66349 2129954 lycopene beta-cyclase (EC 5.5.1.—) - common tobacco AAG21133 10644119 chromoplast-specific lycopene beta-cyclase [Lycopersicon esculentum] CAB92977 8247354 neoxanthin synthase [Solanum tuberosum] CAB93342 8249885 neoxanthin synthase [Lycopersicon esculentum] Q9SEA0 11131528 Capsanthin/capsorubin synthase, chloroplast precursor Q42435 12643508 Capsanthin/capsorubin synthase, chloroplast precursor AAO64977 37730608 lycopene beta cyclase [Haematococcus pluvialis] Q40424 11133011 Lycopene beta cyclase, chloroplast precursor ABB52072 79154940 putative capsanthin-capsorubin synthase [Daucus carota subsp. sativus] AAQ02668 33304511 lycopene cyclase [Setaria italica] CAA54961 840729 putative chromoplastic oxydo-reductase [Capsicum annuum] EAJ62838 44489136 unknown [environmental sequence] YP_401079 81300871 Lycopene cyclase, beta and epsilon [Synechococcus elongatus PCC 7942] YP_172741 56752040 lycopene cyclase [Synechococcus elongatus PCC 6301] ZP_011 . . . 88808972 lycopene beta cyclase [Synechococcus sp. WH 7805] EAK50052 44615956 unknown [environmental sequence] NP_892751 33861190 putative lycopene epsilon cyclase [Prochlorococcus marinus subsp. pastoris str. CCMP1986] NP_875182 33240240 Lycopene epsilon cyclase [Prochlorococcus marinus subsp. marinus str. CCMP1375] YP_382237 78213458 Lycopene cyclase, beta and epsilon [Synechococcus sp. CC9605] YP_397130 78779018 Lycopene cyclase, beta and epsilon [Prochlorococcus marinus str. MIT 9312] NP_896821 33865262 lycopene beta cyclase [Synechococcus sp. WH 8102] YP_397570 78779458 Lycopene cyclase, beta and epsilon [Prochlorococcus marinus str. MIT 9312] ZP_010 . . . 87302144 lycopene cyclase [Synechococcus sp. WH 5701] EAK17149 44569190 unknown [environmental sequence] YP_291882 72382527 lycopene cyclase, beta and epsilon [Prochlorococcus marinus str. NATL2A] NP_875528 33240586 Lycopene beta cyclase related dehydrogenase [Prochlorococcus marinus subsp. marinus str. CCMP1375] NP_893181 33861620 putative lycopene beta cyclase [Prochlorococcus marinus subsp. pastoris str. CCMP1986] NP_895600 33864040 putative lycopene epsilon cyclase [Prochlorococcus marinus str. MIT 9313] EAI47456 44325573 unknown [environmental sequence] YP_291268 72381913 lycopene cyclase, beta and epsilon [Prochlorococcus marinus str. NATL2A] ZP_010 . . . 84517806 Lycopene beta cyclase related dehydrogenase [Prochlorococcus marinus str. MIT 9211] AAF34191 6970079 lycopene epsilon cyclase [Daucus carota] ZP_010 . . . 84518202 Lycopene epsilon cyclase [Prochlorococcus marinus str. MIT 9211] YP_376736 78184301 Lycopene cyclase, beta and epsilon [Synechococcus sp. CC9902] ZP_003 . . . 66796756 Lycopene cyclase, beta and epsilon [Deinococcus geothermalis DSM 11300] NP_894954 33863394 putative lycopene beta cyclase [Prochlorococcus marinus str. MIT 9313] AAT76051 50365502 lycopene cyclase [Citrus clementina] EAK22047 44576122 unknown [environmental sequence] NP_294525 15805827 lycopene cyclase [Deinococcus radiodurans R1]

TABLE 24 Examples of carotenoid glucosyltransferase polypeptides. ACCESSION GI PROTEIN DESCRIPTION AAA21261 148395 CrtX [Pantoea agglomerans] AAN85597 27228291 Zeaxanthin Glucosyl Transferase [Pantoea stewartii] BAB79601 18143446 crtX [Pantoea agglomerans pv. milletiae] AAZ73147 72536082 zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium DC413] AAZ73128 72536060 zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium DC260] AAZ73140 72536074 zeaxanthin glucosyl transferase [Enterobacteriaceae bacterium DC416] Q01330 231911 Zeaxanthin glucosyl transferase ZP_006 . . . 71674312 UDP-glycosyltransferase, MGT [Trichodesmium erythraeum IMS101] NP_439972 16329244 zeaxanthin glucosyl transferase [Synechocystis sp. PCC 6803] EAH29368 44130903 unknown [environmental sequence] ZP_005 . . . 67926135 zeaxanthin glucosyl transferase, hypothetical protein [Crocosphaera watsonii WH 8501] YP_378763 78188425 hypothetical protein Cag_0447 [Chlorobium chlorochromatii CaD3] ZP_005 . . . 68549418 Glycosyl transferase, group 1 [Pelodictyon phaeoclathratiforme BU-1] ZP_010 . . . 85713606 glycosyl transferase, group 1 [Nitrobacter sp. Nb-311A] YP_317171 75674750 glycosyl transferase, group 1 [Nitrobacter winogradskyi Nb- 255] ZP_006 . . . 69929171 Glycosyl transferase, group 1 [Nitrobacter hamburgensis X14] ZP_009 . . . 84500589 hypothetical protein OB2597_11541 [Oceanicola batsensis HTCC2597] ZP_009 . . . 83953176 hypothetical protein NAS141_12746 [Sulfitobacter sp. NAS- 14.1] ZP_009 . . . 83942121 hypothetical protein EE36_07793 [Sulfitobacter sp. EE-36] YP_508020 89052569 glycosyl transferase, group 1 [Jannaschia sp. CCS1] ZP_010 . . . 85704103 hypothetical protein ROS217_13931 [Roseovarius sp. 217] ZP_009 . . . 83370850 probable glycosyltransferase [Rhodobacter sphaeroides ATCC 17025] ZP_006 . . . 69934465 Glycosyl transferase, group 1 [Paracoccus denitrificans PD1222] ZP_009 . . . 83949880 probable glycosyltransferase [Roseovarius nubinhibens ISM] YP_376237 78183803 putative glycosyltransferase [Synechococcus sp. CC9902] YP_376129 78183695 probable glycosyltransferase [Synechococcus sp. CC9902] YP_374296 78186253 hypothetical protein Plut_0365 [Pelodictyon luteolum DSM 273] ZP_010 . . . 87301651 Putative glycosyltransferase [Synechococcus sp. WH 5701] ZP_011 . . . 88809938 Putative glycosyltransferase [Synechococcus sp. WH 7805] BAE47471 78483937 carotenoid glucosyltransferase [Paracoccus sp. N81106] ZP_010 . . . 87303273 probable glycosyltransferase [Synechococcus sp. WH 5701] YP_376127 78183693 probable glycosyltransferase [Synechococcus sp. CC9902] YP_501334 88196509 hypothetical protein SAOUHSC_02880 [Staphylococcus aureus subsp. aureus NCTC 8325] YP_187370 57652300 glycosyl transferase, group 2 family protein [Staphylococcus aureus subsp. aureus COL] CAA66627 1340131u nnamed protein product [Staphylococcus aureus] YP_041987 49484763 putative glycosyl transferase [Staphylococcus aureus subsp. aureus MRSA252] YP_417885 82752144 hypothetical protein SAB2436c [Staphylococcus aureus RF122] YP_252404 70725490 hypothetical protein SH0489 [Staphylococcus haemolyticus JCSC1435] NP_693379 23099913 hypothetical protein OB2458 [Oceanobacillus iheyensis HTE831] ZP_008 . . . 82501285 conserved hypothetical protein [Caldicellulosiruptor saccharolyticus DSM 8903] ZP_010 . . . 87303565 hypothetical protein WH5701_09900 [Synechococcus sp. WH 5701]

TABLE 25 Examples of acyl CoA:diacyglycerol acyltransferase (DGAT) polypeptides. ACCESSION GI PROTEIN DESCRIPTION XP_957022 85082953 hypothetical protein [Neurospora crassa N150] XP_386864 46124621 hypothetical protein FG06688.1 [Gibberella zeae PH-1] XP_755172 71000982 diacylglycerol O-acyltransferase DGAT [Aspergillus fumigatus Af293] XP_663763 67539978 hypothetical protein AN6159.2 [Aspergillus nidulans FGSC A4] BAE65302 83775179 unnamed protein product [Aspergillus oryzae] XP_502557 50550169 hypothetical protein [Yarrowia lipolytica] AAS78662 56199782 diacylglycerol acyltransferase [Glycine max] ABB84383 82582915 diacylglycerol acyltransferase [Jatropha curcas] AAV31083 54145459 1,2-diacyl-sn-glycerol:acyl-CoA acyltransferase [Euonymus alatus] AAG23696 10803053 diacylglycerol acyltransferase [Perilla frutescens] AAF64065 7576941 putative diacylglycerol acyltransferase [Brassica napus] AAS01606 41387497 acyl-CoA:diacylglycerol acyltransferase 1 [Olea europaea] AAT73629 50299542 acyl CoA:diacylglycerol acyltransferase [Glycine max] AAM03340 67043496 putative diacylglycerol acyltransferase [Tropaeolum majus] XP_645633 66824557 hypothetical protein DDB0202877 [Dictyostelium discoideum] AAF19345 6625653 diacylglycerol acylCoA acyltransferase [Nicotiana tabacum] AAY40785 63376239 diacylglycerol acyltransferase DGAT2 [Brassica juncea] AAW47581 57231736 diacylglycerol acyltransferase [Oryza sativa (japonica cultivar-group)] AAR11479 38146080 diacylglycerol acyltransferase [Ricinus communis] AAY40784 63376226 diacylglycerol acyltransferase DGAT1 [Brassica juncea] AAP68322 31711932 At2g19450 [Arabidopsis thaliana] AAW51456 57545061 diacylglycerol acyltransferase [Lotus corniculatus var. japonicus] AAD45536 5579408 putative diacylglycerol acyltransferase [Brassica napus] BAD53762 53791817 putative acyl-CoA:diacylglycerol acyltransferase [Oryza sativa (japonica cultivar-group)] NP_956024 41054343 hypothetical protein LOC325875 [Danio rerio] AAL49962 18642598 diacylglycerol acyltransferase 1 [Bos taurus] XP_930884 89028385 similar to Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase) (ACAT-related gene) [Homo sapiens] NP_777118 27819636 diacylglycerol O-acyltransferase 1 [Bos taurus] Q9GMF1 18202926 Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase) NP_036211 6912332 diacylglycerol O-acyltransferase 1 [Homo sapiens] AAH06263 34782946 DGAT1 protein [Homo sapiens] XP_780515 72006039 similar to Diacylglycerol O-acyltransferase 1 [Strongylocentrotus purpuratus] AAD40881 5225382 putative diacylglycerol acyltransferase [Brassica napus] XP_539214 73974769 similar to Diacylglycerol O-acyltransferase 1 (ACAT related gene product 1) isoform 1 [Canis familiaris] AAZ22403 71063860 diacylglycerol O-acyltransferase 1 [Bubalus bubalis] NP_999216 47522918 diacylglycerol acyltransferase [Sus scrofa] NP_001 . . . 50539976 hypothetical protein LOC436731 [Danio rerio] XP_849176 73974767 similar to Diacylglycerol O-acyltransferase 1 (ACAT related gene product 1) isoform 2 [Canis familiaris] NP_505828 71997360 H19N07.4 [Caenorhabditis elegans] AAF82410 9049538 diacylglycerol acyltransferase [Caenorhabditis elegans] CAE75170 39591950 Hypothetical protein CBG23107 [Caenorhabditis briggsae] XP_626337 66358318 diacylglycerol acyltransferase 1 [Cryptosporidium parvum Iowa II] XP_668402 67624239 acyl-CoA:diacylglycerol acyltransferase 1-related enzyme [Cryptosporidium hominis TU502] AAP94208 33113253 acyl-CoA:diacylglycerol acyltransferase 1-related enzyme [Toxoplasma gondii] AAP94209 33113255 acyl-CoA:diacylglycerol acyltransferase 1-related enzyme [Toxoplasma gondii] XP_579557 62652535 PREDICTED: diacylglycerol O-acyltransferase 1 [Rattus norvegicus] BAC66171 29170489 diacylglycerol acyltransferase [Mus musculus] Q9ERM3 18202872 Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase) AAL78366 18698659 acyl coenzyme A:diacylglycerol acyltransferase [Drosophila melanogaster] NP_995724 45552403 CG31991-PD, isoform D [Drosophila melanogaster] NP_724017 24584734 CG31991-PC, isoform C [Drosophila melanogaster] XP_858062 73974765 similar to Diacylglycerol O-acyltransferase 1 (ACAT related gene product 1) isoform 3 [Canis familiaris] XP_728984 82915156 hypothetical protein PY01256 [Plasmodium yoelii yoelii str. 17XNL] CAG11944 47225461 unnamed protein product [Tetraodon nigroviridis] BAD27526 50199438 acyl-CoA:diacylglycerol acyltransferase [eukaryotic synthetic construct] XP_317656 31226099 ENSANGP00000002281 [Anopheles gambiae str. PEST] AAV59457 55733950 putative diacylglycerol acyltransferase [Oryza sativa (japonica cultivar-group)] EAL33593 54644853 GA16599-PA [Drosophila pseudoobscura] XP_678753 68073677 diacylglycerol O-acyltransferase [Plasmodium berghei strain ANKA] XP_520014 55631434 PREDICTED: similar to Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase) [Pan troglodytes] CAG10815 47219451 unnamed protein product [Tetraodon nigroviridis] XP_624754 66522700 PREDICTED: similar to ENSANGP00000002281 [Apis mellifera] CAC69884 15620769 diacylglycerol acyltransferase I [Rattus norvegicus] XP_686181 68363630 PREDICTED: similar to Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase) [Danio rerio] XP_734008 70921323 diacylglycerol O-acyltransferase [Plasmodium chabaudi chabaudi] XP_673128 68062248 hypothetical protein PB300300.00.0 [Plasmodium berghei strain ANKA] AAS72376 45642963 acyl-CoA:cholesterol acyltransferase beta [Toxoplasma gondii] AAS72375 45642961 acyl-CoA:cholesterol acyltransferase alpha [Toxoplasma gondii] NP_586145 19074639 STEROL O-ACYLTRANSFERASE [Encephalitozoon cuniculi GB-M1] XP_640280 66812202 hypothetical protein DDB0205259 [Dictyostelium discoideum] AAY40783 63376221 diacylglycerol acyltransferase [Brassica juncea] XP_765774 71032265 diacylglycerol O-acyltransferase [Theileria parva strain Muguga] Q876L2 34582301 Sterol O-acyltransferase 2 (Sterol-ester synthase 2) XP_571260 58268208 sterol O-acyltransferase [Cryptococcus neoformans var. neoformans JEC21] EAL20032 50257323 hypothetical protein CNBF3580 [Cryptococcus neoformans var. neoformans B-3501A] XP_954478 84999514 acyl transferase [Theileria annulata strain Ankara] XP_505086 50555355 hypothetical protein [Yarrowia lipolytica] NP_588558 19076058 hypothetical protein SPCP1E11.05c [Schizosaccharomyces pombe 972h-] AAC49441 1389739 acyl-CoA:sterol acyltransferase NP_014416 6324346 Acyl-CoA:sterol acyltransferase, isozyme of Are1p; Are2p [Saccharomyces cerevisiae] XP_750354 70991010 sterol o-acyltransferase APE2 [Aspergillus fumigatus Af293] XP_382192 46110268 hypothetical protein FG02016.1 [Gibberella zeae PH-1] BAE54934 83764790 unnamed protein product [Aspergillus oryzae] XP_885914 76617939 similar to Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) isoform 2 [Bos taurus] XP_591251 76617937 similar to Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) isoform 1 [Bos taurus] BAC00846 21392392 AcylCoA:Cholesterol Acyltransferase 2 [Rattus norvegicus] NP_649816 28571583 CG8112-PA [Drosophila melanogaster] NP_666176 22122547 sterol O-acyltransferase 2 [Mus musculus] O88908 18202245 Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) XP_761502 71022545 hypothetical protein UM05355.1 [Ustilago maydis 521] NP_714950 40254723 sterol O-acyltransferase 2 [Rattus norvegicus] EAQ86094 88178626 hypothetical protein CHGG_07347 [Chaetomium globosum CBS 148.51] XP_461395 50425599 hypothetical protein DEHA0F25652g [Debaryomyces hansenii CBS767] XP_661812 67527926 hypothetical protein AN4208.2 [Aspergillus nidulans FGSC A4] AAH96091 64654094 Sterol O-acyltransferase 2 [Homo sapiens] O75908 18202149 Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) AAH96090 64652990 Sterol O-acyltransferase 2 [Homo sapiens] AAK48829 13898623 acyl coenzyme A: cholesterol acyltransferase-2 [Homo sapiens] XP_543637 73996435 PREDICTED: similar to sterol O-acyltransferase 2 [Canis familiaris] O77759 18202176 Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) AAO32474 28564191 ARE2 [Saccharomyces castellii] XP_323485 32405744 hypothetical protein [Neurospora crassa] NP_982606 45184888 AAR065Cp [Eremothecium gossypii] NP_593708 19114620 hypothetical protein SPAC13G7.06 [Schizosaccharomyces pombe 972h-] AAO32554 28564940 ARE2 [Saccharomyces kluyveri] EAL28962 54639560 GA20833-PA [Drosophila pseudoobscura] XP_449806 50294790 hypothetical protein CAGL0M10571g [Candida glabrata CBS138] NP_033256 84619697 sterol O-acyltransferase 1 [Mus musculus] Q61263 18202591 Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) BAC34925 26342537 unnamed protein product [Mus musculus] XP_452607 50305295 unnamed protein product [Kluyveromyces lactis] NP_001 . . . 77735363 hypothetical protein LOC504287 [Bos taurus] Q60457 18202585 Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) XP_320321 58393811 ENSANGP00000016512 [Anopheles gambiae str. PEST] XP_320320 58393809 ENSANGP00000016486 [Anopheles gambiae str. PEST] O70536 18202126 Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) XP_714776 68482533 acyl-CoA cholesterol acyltransferase [Candida albicans SC5314] P84285 56404462 Sterol O-acyltransferase 2 (Sterol-ester synthase) (ASAT) AAH77916 50416229 Soat1-prov protein [Xenopus laevis] XP_692855 68364838 PREDICTED: similar to Soat1-prov protein [Danio rerio] CAI13574 55960156 sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1 [Homo sapiens] AAL56227 18028942 cholesterol acyltransferase 1 [Gorilla gorilla] AAL56228 18028944 cholesterol acyltransferase 1 [Pongo pygmaeus] AAC37532 4878022 acyl-coenzyme A: cholesterol acyltransferase [Homo sapiens] 2201440A 1585676 acyl-CoA/cholesterol acyltransferase Q876L3 34582302 Sterol O-acyltransferase 1 (Sterol-ester synthase 1) BAE01048 67969393 unnamed protein product [Macaca fascicularis] XP_514030 55588858 PREDICTED: hypothetical protein XP_514030 [Pan troglodytes] XP_547445 73961286 similar to Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) [Canis familiaris] EAQ84619 88177151 hypothetical protein CHGG_08633 [Chaetomium globosum CBS 148.51] O77761 18202178 Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) XP_422267 50751122 PREDICTED: similar to Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) [Gallus gallus] XP_693284 68392980 PREDICTED: similar to Sterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) [Danio rerio] AAT92940 51013293 YCR048W [Saccharomyces cerevisiae] XP_956576 85080625 hypothetical protein [Neurospora crassa N150] XP_624691 66564061 PREDICTED: similar to ENSANGP00000016486 [Apis mellifera] CAF96514 47222847 unnamed protein product [Tetraodon nigroviridis] XP_788209 72085563 PREDICTED: similar to sterol O-acyltransferase 1 [Strongylocentrotus purpuratus] XP_445307 50285757 unnamed protein product [Candida glabrata] CAE70002 39596364 Hypothetical protein CBG16409 [Caenorhabditis briggsae] CAG07990 47225647 unnamed protein product [Tetraodon nigroviridis] NP_510623 17549960 B0395.2 [Caenorhabditis elegans] AAX28331 76157393 SJCHGC04421 protein [Schistosoma japonicum] CAI96158 66347204 Diacylglycerol O-acyltransferase [Bubalus bubalis] XP_390039 46136695 hypothetical protein FG09863.1 [Gibberella zeae PH-1] XP_643169 66819019 hypothetical protein DDB0203882 [Dictyostelium discoideum] AAO53095 28850306 hypothetical protein [Dictyostelium discoideum] AAB06959 1515472 acyl-CoA:cholesterol acyltransferase [Oryctolagus cuniculus] NP_945619 39933343 putative alginate o-acetyltransferase AlgI [Rhodopseudomonas palustris CGA009] ZP_008 . . . 77691302 Membrane bound O-acyl transferase, MBOAT [Rhodopseudomonas palustris BisB5] XP_465546 50908115 putative wax synthase [Oryza sativa (japonica cultivar- group)]

TABLE 29 Examples of Prenyldiphosphate synthase polypeptides Accession GI Description 29A: Bacteria Proteins that require a mitochondrial targeting sequence ZP_009 . . . 83373595 Trans-hexaprenyltranstransferase [Rhodobacter sphaeroides ATCC 17029] ZP_009 . . . 83371280 Trans-hexaprenyltranstransferase [Rhodobacter sphaeroides ATCC 17025] CAD24417 20429105 decaprenyl diphosphate synthase [Paracoccus zeaxanthinifaciens] ZP_010 . . . 85705714 Geranylgeranyl pyrophosphate synthase/Polyprenyl synthetase [Roseovarius sp. 217] ZP_010 . . . 84515724 decaprenyl diphosphate synthase [Loktanella vestfoldensis SKA53] YP_165582 56695234 decaprenyl diphosphate synthase [Silicibacter pomeroyi DSS-3] ZP_010 . . . 86139019 decaprenyl diphosphate synthase [Roseobacter sp. MED193] ZP_009 . . . 83941379 decaprenyl diphosphate synthase [Sulfitobacter sp. EE-36] ZP_009 . . . 83854856 decaprenyl diphosphate synthase [Sulfitobacter sp. NAS-14.1] ZP_006 . . . 69299873 Farnesyltranstransferase [Silicibacter sp. TM1040] ZP_010 . . . 84683979 Geranylgeranyl pyrophosphate synthase/Polyprenyl synthetase [Rhodobacterales bacterium HTCC2654] ZP_009 . . . 84500217 decaprenyl diphosphate synthase [Oceanicola batsensis HTCC2597] ZP_009 . . . 83952381 decaprenyl diphosphate synthase [Roseovarius nubinhibens ISM] ZP_006 . . . 69937106 Trans-hexaprenyltranstransferase [Paracoccus denitrificans PD1222] ZP_005 . . . 68180845 Trans-hexaprenyltranstransferase [Jannaschia sp. CCS1] ZP_008 . . . 78495595 Polyprenyl synthetase [Rhodopseudomonas palustris BisB18] AAY82368 67866738 decaprenyl diphosphate synthase [Agrobacterium tumefaciens] NP_353656 15887975 hypothetical protein AGR_C_1125 [Agrobacterium tumefaciens str. C58] ZP_008 . . . 77688465 Farnesyltranstransferase [Rhodopseudomonas palustris BisB5] NP_531334 17934544 octaprenyl-diphosphate synthase [Agrobacterium tumefaciens str. C58] YP_484709 86748213 Farnesyltranstransferase [Rhodopseudomonas palustris HaA2] AAP56240 37903500 decaprenyl diphosphate synthase [Agrobacterium tumefaciens] YP_192388 58040424 Decaprenyl diphosphate synthase [Gluconobacter oxydans 621H] 29B: Subunit 1 - Proteins that contain mitochondrial targeting sequence T43193 11279237 trans-pentaprenyltranstransferase homolog - fission yeast (Schizosaccharomyces pombe) AAD28559 4732024 trans-prenyltransferase [Homo sapiens] AAI07275 78070698 Trans-prenyltransferase [Mus musculus] BAE48216 81157931 subunit 1 of decaprenyl diphosphate synthase [Homo sapiens] AAH49211 29165656 PDSS1 protein [Homo sapiens] Q33DR2 85700953 Decaprenyl-diphosphate synthase subunit 1 (Solanesyl-diphosphate synthase subunit 1) (Trans-prenyltransferase) XP_507706 55633583 PREDICTED: similar to TPRT protein [Pan troglodytes] XP_586717 76632198 PREDICTED: similar to trans-prenyltransferase [Bos taurus] XP_849908 73948851 PREDICTED: similar to trans-prenyltransferase [Canis familiaris] 29C: Subunit 2 - Proteins that contain mitochondrial targeting sequence O13851 60389474 Decaprenyl-diphosphate synthase subunit 2 (Decaprenyl pyrophosphate synthetase subunit 2) BAE48218 81157935 subunit 2 of solanesyl diphosphate synthase [Mus musculus] BAE48217 81157933 subunit 2 of decaprenyl diphosphate synthase [Homo sapiens]

TABLE 30 Examples of PHB-Polyprenyltransferase polypeptides GI PROTEIN DESCRIPTION 51013645 YNR041C [Saccharomyces cerevisiae] 50285815 unnamed protein product [Candida glabrata] 50311051 unnamed protein product [Kluyveromyces lactis] 45200866 AGL231Wp [Eremothecium gossypii] 50555263 hypothetical protein [Yarrowia lipolytica] 68473193 para-hydroxybenzoate: polyprenyl transferase [Candida albicans SC5314] 50410039 hypothetical protein DEHA0A14212g [Debaryomyces hansenii CBS767] 83769349 unnamed protein product [Aspergillus oryzae] 70994900 para-hydroxybenzoate-polyprenyltransferase precursor [Aspergillus fumigatus Af293] 19114131 hypothetical protein SPAC56F8.04c [Schizosaccharomyces pombe 972h-] 39973573 hypothetical protein MG01067.4 [Magnaporthe grisea 70-15] 85078920 protein related to para-hydroxybenzoate polyprenyltransferase precursor [Neurospora crassa N150] 76660839 PREDICTED: similar to para-hydroxybenzoate- polyprenyltransferase, mitochondrial [Bos taurus] 52138578 para-hydroxybenzoate-polyprenyltransferase, mitochondrial [Homo sapiens] 18088424 COQ2 protein [Homo sapiens] 47221448 unnamed protein product [Tetraodon nigroviridis] 58385249 ENSANGP00000012220 [Anopheles gambiae str. PEST] 50746583 PREDICTED: similar to hypothetical protein CL640 [Gallus gallus] 54638587 GA21912-PA [Drosophila pseudoobscura] 21355567 CG9613-PA [Drosophila melanogaster] 71005862 hypothetical protein UM01450.1 [Ustilago maydis 521]

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: 

1-63. (canceled)
 64. A genetically engineered fungus characterized by: i. lipid accumulation to at least about 20% of its dry cell weight; ii. production of at least one carotenoid as a result of genetic engineering; and iii. accumulation of the at least one carotenoid to at least about 1% of its dry cell weight.
 65. The genetically engineered fungus of claim 64, wherein the lipid accumulation to at least about 20% of its dry cell weight occurs as a result of genetic engineering.
 66. The genetically engineered fungus of claim 64, wherein the fungus is selected from a genera group comprising Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon, and Yarrowia.
 67. The genetically engineered fungus of claim 66, wherein the fungus is selected from a species group consisting of Blakeslea trispora, Candida pulcherrima, C. revkaufi, C. tropicalis, Cryptococcus curvatus, Cunninghamella echinulata, C. elegans, C. japonica, Lipomyces starkeyi, L. lipoferus, Mortierella alpina, M. isabellina, M. ramanniana, M. vinacea, Mucor circinelloides, Phycomyces blakesleanus, Pythium irregulare, Rhodosporidium toruloides, Rhodotorula glutin is, R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, Trichosporon pullans, T. cutaneum, and Yarrowia lipolytica.
 68. The genetically engineered fungus of claim 67, wherein the fungus is Blakeslea trispora.
 69. The genetically engineered yeast of claim 64, wherein the fungus is a yeast.
 70. The genetically engineered yeast of claim 69, wherein the at least one carotenoid is β-carotene.
 71. The genetically engineered yeast of claim 69, wherein the yeast grows as a single cell
 72. The genetically engineered fungus of claim 64, wherein the at least one carotenoid is accumulated in cytoplasmic oil bodies.
 73. The genetically engineered fungus of claim 64, wherein the carotenoid is selected from the group consisting of astaxanthin, β-carotene, canthaxanthin, zeaxanthin, lutein, lycopene, and combinations thereof.
 74. The genetically engineered fungus of claim 73, wherein the carotenoid is astaxanthin.
 75. The genetically engineered fungus of claim 73, wherein the carotenoid is β-carotene.
 76. The genetically engineered fungus of claim 73, wherein the carotenoid is canthaxanthin.
 77. The genetically engineered fungus of claim 73, wherein the carotenoid is zeaxanthin.
 78. The genetically engineered fungus of claim 64, wherein the genetic engineering alters the expression or activity of a carotenogenic polypeptide selected from the group consisting of isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, isoprenoid biosynthesis competitor polypeptides, and combinations thereof.
 79. The genetically engineered fungus of claim 78, wherein the isoprenoid biosynthesis polypeptide is selected from the group consisting acetoacetyl-CoA thiolase polypeptide, HMG-CoA synthase polypeptide, HMG-CoA reductase polypeptide, mevalonate kinase polypeptide, phosphomevalonate kinase polypeptide, mevalonate pyrophosphate decarboxylase polypeptide, IPP isomerase polypeptide, FPP synthase polypeptide, and GGPP synthase polypeptide.
 80. The genetically engineered fungus of claim 78, wherein the carotenoid biosynthesis polypeptide is selected from the group consisting of: phytoene synthase polypeptide, phytoene dehydrogenase polypeptide, lycopene cyclase polypeptide, carotenoid ketolase polypeptide, carotenoid hydroxylase polypeptide, astaxanthin synthase polypeptide, carotenoid epsilon hydroxylase polypeptide, carotenoid glucosyltransferase polypeptide, Iycopene cyclase (beta and epsilon subunits) polypeptides, and acyl CoA:diacyglycerol acyltransferase polypeptide.
 81. The genetically engineered fungus of claim 78, wherein the isoprenoid biosynthesis competitor polypeptide is selected from the group consisting of squalene synthase polypeptide, prenyldiphosphate synthase and PHB polyprenyltransferase.
 82. The genetically engineered fungus of claim 78, wherein the carotenogenic polypeptide confers to the yeast the ability to produce the at least one carotenoid to a level at least about 1% of its dry cell weight or confers to the yeast the ability to produce at least one carotenoid which the yeast does not naturally produce.
 83. The genetically engineered fungus of claim 78, wherein the fungus comprises at least one modification selected from the group consisting of: i. expression of truncated HMG-CoA reductase; ii. heterologous expression of carotene hydroxylase and carotene ketolase; iii. complete or partial inactivation of a gene encoding squalene synthase; and iv. combinations thereof.
 84. The genetically engineered fungus of claim 64, further characterized by an oleaginic modification.
 85. The genetically engineered fungus of claim 84, wherein the oleaginic modification confers upon the fungus an ability to accumulate lipid to at least about 20% of its dry cell weight.
 86. The genetically engineered fungus of claim 84, wherein the oleaginic modification alters expression or activity of at least one oleaginic polypeptide.
 87. The genetically engineered fungus of claim 86, wherein the at least one oleaginic polypeptide is selected from the group consisting of acetyl-CoA carboxylase polypeptide, pyruvate decarboxylase polypeptide, isocitrate dehydrogenase polypeptide, ATP-citrate lyase polypeptide, malic enzyme polypeptide, AMP deaminase polypeptide, and combinations thereof.
 88. The genetically engineered fungus of claim 84, wherein the fungus is selected from a genera group consisting of Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia).
 89. The genetically engineered fungus of claim 84, wherein the fungus is selected from a species group consisting of Aspergillus nidulans, A. niger, A. terreus, Botrytis cinerea, Cercospora nicotianae, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, K. lactis, Neurospora crassa, Pichia pastoris, Puccinia distincta, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, and Xanthophyllomyces dendrorhous (Phaffia rhodozyma).
 90. The genetically engineered fungus of claim 89, wherein the fungus is Saccharomyces cerevisiae.
 91. An engineered fungus selected from the group consisting of Aspergillus nidulans, A. niger, A. terreus, Botrytis cinerea, Cercospora nicotianae, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis, K. lactis, Neurospora crassa, Pichia pastoris, Puccinia distincta, Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, and Xanthophyllomyces dendrorhous (Phaffia rhodozyma), which engineered fungus has been genetically engineered to contain carotenogenic modifications sufficient to permit the engineered fungus to produce at least one carotenoid that it does not naturally produce.
 92. The engineered fungus of claim 91, wherein the yeast accumulates at least one carotenoid to a level at least about 1% of its dry cell weight.
 93. The engineered fungus of claim 91, further comprising an oleaginic modification that confers the ability to accumulate lipids to at least about 20% of its dry cell weight.
 94. The engineered fungus of claim 91, wherein the carotenoid is astaxanthin.
 95. The engineered fungus of claim 91, wherein the carotenoid is β-carotene.
 96. The engineered fungus of claim 91, wherein the carotenogenic modifications comprise the introduction of genes encoding carotenogenic polypeptides selected from the group consisting of isoprenoid biosynthesis polypeptides, carotenoid biosynthesis polypeptides, isoprenoid biosynthesis competitor polypeptides, and combinations thereof.
 97. A method of producing astaxanthin, the method comprising cultivating a genetically engineered fungus under conditions that allow production of astaxanthin; wherein the genetically engineered fungus is characterized by: i. the fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and ii. as a result of the genetic engineering, the fungus produces astaxanthin to at least about 1% of its dry cell weight.
 98. The method of claim 97, wherein the method comprises a second step of isolating the astaxanthin.
 99. The method of claim 97, wherein the step of isolating comprises fractionating the cultivation medium to obtain at least one carotenoid-enriched fraction.
 100. The method of claim 97, wherein the step of cultivating comprises cultivating the fungus under conditions that allow accumulation of the astaxanthin in cytoplasmic oil bodies, and the step of isolating comprises isolating oil derived from the cytoplasmic oil bodies. 